Antibodies that bind to PRO286

ABSTRACT

The invention relates to the identification and isolation of novel DNAs encoding the human Toll proteins PRO285, PRO286, and PRO358, and to methods and means for the recombinant production of these proteins. The invention also concerns antibodies specifically binding the PRO285, or PRO286, or PRO358 Toll protein.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/202,054 filed Dec. 7, 1998, now U.S. Pat. No. 7,696,327 which is aSection 371 application of PCT/US98/21141 filed Oct. 7, 1998, claimingpriority to provisional applications 60/062,250 filed Oct. 17, 1997;60/065,311 filed Nov. 13, 1997; 60/083,322 filed Apr. 28, 1998;60/090,863 filed Jun. 26, 1998; and 09/105,413 filed Jun. 26, 1998, thecontents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to the identification andisolation of novel DNAs designated herein as DNA40021, DNA42663 andDNA47361, and to the recombinant production of novel human Tollhomologues (designated as PRO285, PRO286 and PRO358, respectively)encoded by said DNAs.

BACKGROUND OF THE INVENTION

Membrane-bound proteins and receptors can play an important role in theformation, differentiation and maintenance of multicellular organisms.The fate of many individual cells, e.g., proliferation, migration,differentiation, or interaction with other cells, is typically governedby information received from other cells and/or the immediateenvironment. This information is often transmitted by secretedpolypeptides (for instance, mitogenic factors, survival factors,cytotoxic factors, differentiation factors, neuropeptides, and hormones)which are, in turn, received and interpreted by diverse cell receptorsor membrane-bound proteins. Such membrane-bound proteins and cellreceptors include, but are not limited to, cytokine receptors, receptorkinases, receptor phosphatases, receptors involved in cell-cellinteractions, and cellular adhesin molecules like selectins andintegrins. For instance, transduction of signals that regulate cellgrowth and differentiation is regulated in part by phosphorylation ofvarious cellular proteins. Protein tyrosine kinases, enzymes thatcatalyze that process, can also act as growth factor receptors. Examplesinclude fibroblast growth factor receptor and nerve growth factorreceptor.

Membrane-bound proteins and receptor molecules have various industrialapplications, including as pharmaceutical and diagnostic agents.Receptor immunoadhesins, for instance, can be employed as therapeuticagents to block receptor-ligand interaction. The membrane-bound proteinscan also be employed for screening of potential peptide or smallmolecule inhibitors of the relevant receptor/ligand interaction.

Efforts are being undertaken by both industry and academia to identifynew, native receptor proteins. Many efforts are focused on the screeningof mammalian recombinant DNA libraries to identify the coding sequencesfor novel receptor proteins.

The cloning of the Toll gene of Drosophila, a maternal effect gene thatplays a central role in the establishment of the embryonicdorsal-ventral pattern, has been reported by Hashimoto et al., Cell 52,269-279 (1988). The Drosophila Toll gene encodes an integral membraneprotein with an extracytoplasmic domain of 803 amino acids and acytoplasmic domain of 269 amino acids. The extracytoplasmic domain has apotential membrane-spanning segment, and contains multiple copies of aleucine-rich segment, a structural motif found in many transmembraneproteins. The Toll protein controls dorsal-ventral patterning inDrosophila embryos and activates the transcription factor Dorsal uponbinding to its ligand Spätzle. (Morisato and Anderson, Cell 76, 677-688(1994).) In adult Drosophila, the Toll/Dorsal signaling pathwayparticipates in the anti-fungal immune response. (Lenaitre et al., Cell86, 973-983 (1996).)

A human homologue of the Drosophila Toll protein has been described byMedzhitov et al., Nature 388, 394-397 (1997). This human Toll, just asDrosophila Toll, is a type I transmembrane protein, with anextracellular domain consisting of 21 tandemly repeated leucine-richmotifs (leucine-rich region—LRR), separated by a non-LRR region, and acytoplasmic domain homologous to the cytoplasmic domain of the humaninterleukin-1 (IL-1) receptor. A constitutively active mutant of thehuman Toll transfected into human cell lines was shown to be able toinduce the activation of NF-κB and the expression of NF-κB-controlledgenes for the inflammatory cytokines IL-1, IL-6 and IL-8, as well as theexpression of the constimulatory molecule B7.1, which is required forthe activation of native T cells. It has been suggested that Tollfunctions in vertebrates as a non-clonal receptor of the immune system,which can induce signals for activating both an innate and an adaptiveimmune response in vertebrates. The human Toll gene reported byMedzhitov et al., supra was most strongly expressed in spleen andperipheral blood leukocytes (PBL), and the authors suggested that itsexpression in other tissues may be due to the presence of macrophagesand dendritic cells, in which it could act as an early-warning systemfor infection. The public GenBank database contains the following Tollsequences: Toll1 (DNAX# HSU88540-1, which is identical with the randomsequenced full-length cDNA #HUMRSC786-1); Toll2 (DNAX# HSU88878-1);Toll3 (DNAX# HSU88879-1); and Toll4 (DNAX# HSU88880-1, which isidentical with the DNA sequence reported by Medzhitov et al., supra). Apartial Toll sequence (Toll5) is available from GenBank under DNAX#HSU88881-1.

Further human homologues of the Drosophila Toll protein, designated asToll-like receptors (huTLRs1-5) were recently cloned and shown to mirrorthe topographic structure of the Drosophila counterpart (Rock et al.,Proc. Natl. Acad. Sci. USA 95, 588-593 [1998]). Overexpression of aconstitutively active mutant of one human TLR (Toll-proteinhomologue-Medzhitov et al., supra; TLR4—Rock et al., supra) leads to theactivation of NF-κB and induction of the inflammatory cytokines andconstimulatory molecules. Medzhitov et al., supra.

SUMMARY OF THE INVENTION

Applicants have identified three novel cDNA clones that encode novelhuman Toll polypeptides, designated in the present application as PRO285(encoded by DNA40021), PRO286 (encoded by DNA42663), and PRO358 (encodedby DNA47361).

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising a DNA encoding a polypeptide having at least about80% sequence identity, preferably at least about 85% sequence identity,more preferably at least about 90% sequence identity, most preferably atleast about 95% sequence identity to (a) a DNA molecule encoding aPRO285 polypeptide having amino acid residues 27 to 839 of FIG. 1 (SEQID NO:1); or (b) to a DNA molecule encoding a PRO286 polypeptide havingamino acid residues 27 to 825 of FIG. 3 (SEQ ID NO:3), or (c) to a DNAmolecule encoding a PRO358 polypeptide having amino acids 20 to 575 ofFIGS. 12A-C (SEQ ID NO: 13), or (d) the complement of the DNA moleculeof (a), (b), or (c). The complementary DNA molecule preferably remainsstably bound to such encoding nucleic acid sequence under at leastmoderate, and optionally, under high stringency conditions.

In a further embodiment, the isolated nucleic acid molecule comprises apolynucleotide that has at least about 90%, preferably at least about95% sequence identity with a polynucleotide encoding a polypeptidecomprising the sequence of amino acids 1 to 839 of FIG. 1 (SEQ ID NO:1);or at least about 90%, preferably at least about 95% sequence identitywith a polynucleotide encoding a polypeptide comprising the sequence ofamino acids 1 to 1041 of FIG. 3 (SEQ ID NO: 3); or at least about 90%,preferably at least about 95% sequence identity with a polynucleotideencoding a polypeptide comprising the sequence of amino acids 1 to 811of FIGS. 12A-C (SEQ ID NO: 13).

In a specific embodiment, the invention provides an isolated nucleicacid molecule comprising DNA encoding native or variant PRO285, PRO286,and PRO358 polypeptides, with or without the N-terminal signal sequence,and with or without the transmembrane regions of the respectivefull-length sequences. In one aspect, the isolated nucleic acidcomprises DNA encoding a mature, full-length native PRO285, PRO286, orPRO358 polypeptide having amino acid residues 1 to 1049 of FIG. 1 (SEQID NO: 1), 1 to 1041 of FIG. 3 (SEQ ID NO: 3), and 1 to 811 of FIGS.12A-C (SEQ ID NO: 13), or is complementary to such encoding nucleic acidsequence. In another aspect, the invention concerns an isolated nucleicacid molecule that comprises DNA encoding a native PRO285, PRO286, orPRO358 polypeptide without an N-terminal signal sequence, or iscomplementary to such encoding nucleic acid sequence. In yet anotherembodiment, the invention concerns nucleic acid encodingtransmembrane-domain deleted or inactivated forms of the full-lengthnative PRO285, PRO286 and PRO358 proteins.

In another aspect, the invention concerns an isolated nucleic acidmolecule encoding a PRO285, PRO286 or PRO358 polypeptide comprising DNAhybridizing to the complement of the nucleic acid between about residues85 and about 3283 inclusive, of FIG. 2 (SEQ ID NO: 2), or to thecomplement of the nucleic acid between about residues 57 and about 4199,inclusive, of FIG. 4 (SEQ ID NO: 4), or to the complement of the nucleicacid between about residues 111 and about 2544 of FIGS. 13A-B (SEQ IDNO: 14). Preferably, hybridization occurs under stringent hybridizationand wash conditions.

In another aspect, the invention concerns an isolated nucleic acidmolecule comprising (a) DNA encoding a polypeptide scoring at leastabout 80% positives, preferably at least about 85% positives, morepreferably at least about 90% positives, most preferably at least about95% positives when compared with the amino acid sequence of residues 1to 1049, inclusive of FIG. 1 (SEQ ID NO:1), or amino acid residues 1 to1041, inclusive of FIG. 3 (SEQ ID NO: 3), or amino acid residues 1 to811, inclusive of FIGS. 12A-C (SEQ ID NO: 13, or (b) the complement of aDNA of (a).

In another embodiment, the invention the isolated nucleic acid moleculecomprises the clone (DNA 40021-1154) deposited on Oct. 17, 1997, underATCC number 209389; or the clone (DNA 42663-1154) deposited on Oct. 17,1997, under ATCC number 209386; or the clone (DNA 47361-1249) depositedon Nov. 7, 1997, under ATCC number 209431.

In yet another embodiment, the invention provides a vector comprisingDNA encoding PRO285, PRO286 and PRO358 polypeptides, or their variants.Thus, the vector may comprise any of the isolated nucleic acid moleculeshereinabove defined.

In a specific embodiment, the invention provides a vector comprising apolynucleotide having at least about 80% sequence identity, preferablyat least about 85% sequence identity, more preferably at least about 90%sequence identity, most preferably at least about 95% sequence identitywith a polynucleotide encoding a polypeptide comprising the sequence ofamino acids 20 to 811 of FIGS. 12A-C (SEQ ID NO:13), or the complementof such polynucleotide. In a particular embodiment, the vector comprisesDNA encoding the novel Toll homologue (PRO358), with or without theN-terminal signal sequence (about amino acids 1 to 19), or atransmembrane-domain (about amino acids 576-595) deleted or inactivatedvariant thereof, or the extracellular domain (about amino acids 20 to595) of the mature protein, or a protein comprising any one of thesesequences. A host cell comprising such a vector is also provided. Asimilar embodiment will be apparent for vectors comprisingpolynucleotides encoding the PRO285 and PRO286 Toll homologues, with ourwithout the respective signal sequences and/or transmembrane-domaindeleted or inactivated variants thereof, and specifically, vectorscomprising the extracellular domains of the mature PRO85 and PRO286 Tollhomologues, respectively.

A host cell comprising such a vector is also provided. By way ofexample, the host cells may be CHO cells, E. coli, or yeast.

A process for producing PRO285, PRO286 and PRO358 polypeptides isfurther provided and comprises culturing host cells under conditionssuitable for expression of PRO285, PRO286, and PRO358, respectively, andrecovering PRO285, PRO286, o PRO358 from the cell culture.

In another embodiment, the invention provides isolated PRO285, PRO286and PRO358 polypeptides. In particular, the invention provides isolatednative sequence PRO285 and PRO286 polypeptides, which in one embodiment,include the amino acid sequences comprising residues 1 to 1049 and 1 to1041 of FIGS. 1 and 3 (SEQ ID NOs:1 and 3), respectively. The inventionalso provides for variants of the PRO285 and PRO286 polypeptides whichare encoded by any of the isolated nucleic acid molecules hereinabovedefined. Specific variants include, but are not limited to, deletion(truncated) variants of the full-length native sequence PRO285 andPRO286 polypeptides which lack the respective N-terminal signalsequences and/or have their respective transmembrane and/or cytoplasmicdomains deleted or inactivated. The invention further provides anisolated native sequence PRO358 polypeptide, or variants thereof. Inparticular, the invention provides an isolated native sequence PRO358polypeptide, which in certain embodiments, includes the amino acidsequence comprising residues 20 to 575, or 20 to 811, or 1 to 811 ofFIGS. 12A-C (SEQ ID NO: 13).

In a further aspect, the invention concerns an isolated PRO285, PRO286or PRO358 polypeptide, comprising an amino acid sequence scoring atleast about 80% positives, preferably at least about 85% positives, morepreferably at least about 90% positives, most preferably at least about95% positives when compared with the amino acid sequence of amino acidresidues 1 to 1049, inclusive of FIG. 1 (SEQ ID NO:1), or amino acidresidues 1 to 1041, inclusive of FIG. 3 (SEQ ID NO: 3), or amino acidresidues 1 to 811, inclusive of FIGS. 12A-C (SEQ ID NO: 13).

In a still further aspect, the invention provides a polypeptide producedby (I) hybridizing a test DNA molecule under stringent conditions with(a) a DNA molecule encoding a PRO285, PRO286 or PRO358 polypeptidehaving the sequence of amino acid residues from about 1 to about 1049,inclusive of FIG. 1 (SEQ ID NO:1), or amino acid residues from about 1to about 1041, inclusive of FIG. 3 (SEQ ID NO: 3), or amino acidresidues from about 1 to about 811, inclusive of FIGS. 12A-C (SEQ ID NO:13), or (b) the complement of a DNA molecule of (a), and if the test DNAmolecule has at least about an 80% sequence identity, preferably atleast about an 85% sequence identity, more preferably at least about a90% sequence identity, most preferably at least about a 95% sequenceidentity to (a) or (b), (ii) culturing a host cell comprising the testDNA molecule under conditions suitable for expression of thepolypeptide, and (iii) recovering the polypeptide from the cell culture.

In another embodiment, the invention provides chimeric moleculescomprising PRO285, PRO286, or PRO358 polypeptides fused to aheterologous polypeptide or amino acid sequence. An example of such achimeric molecule comprises a PRO285, PRO286, or PRO358 polypeptidefused to an epitope tag sequence or a Fc region of an immunoglobulin. Anexample of such a chimeric molecule comprises a PRO358 polypeptide(including its signal peptide and/or transmembrane-domain and,optionally, intracellular domain, deleted variants), fused to an epitopetag sequence or a Fc region of an immunoglobulin. In a preferredembodiment, the fusion contains the extracellular domain of PRO358 fusedto an immunoglobulin constant region, comprising at least the CH2 andCH3 domains. Similar specific embodiments exist and are disclosed hereinfor chimeric molecules comprising a PRO285 or PRO286 polypeptide.

In another embodiment, the invention provides an antibody whichspecifically binds to PRO285, PRO286 or PRO358 polypeptides. Optionally,the antibody is a monoclonal antibody. The invention specificallyincludes antibodies with dual specificities, e.g., bispecific antibodiesbinding more than one Toll polypeptide.

In yet another embodiment, the invention concerns agonists andantagonists of the native PRO285, PRO286 and PRO358 polypeptides. In aparticular embodiment, the agonist or antagonist is an anti-PRO285,anti-PRO286 or anti-PRO358 antibody.

In a further embodiment, the invention concerns screening assays toidentify agonists or antagonists of the native PRO285, PRO286 and PRO358polypeptides.

In a still further embodiment, the invention concerns a compositioncomprising a PRO285, PRO286 or PRO358 polypeptide, or an agonist orantagonist as hereinabove defined, in combination with apharmaceutically acceptable carrier.

The invention further concerns a composition comprising an antibodyspecifically binding a PRO285, PRO286 or PRO358 polypeptide, incombination with a pharmaceutically acceptable carrier.

The invention also concerns a method of treating septic shock comprisingadministering to a patient an effective amount of an antagonist of aPRO285, PRO286 or PRO358 polypeptide. In a specific embodiment, theantagonist is a blocking antibody specifically binding a native PRO285,PRO286 or PRO358 polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the derived amino acid sequence of a native sequence humanToll protein, designated PRO285 (SEQ ID NO: 1).

FIG. 2 shows the nucleotide sequence of a native sequence human Tollprotein cDNA designated DNA40021 (SEQ ID NO: 2), which encodes PRO285.

FIG. 3 shows the derived amino acid sequence of a native sequence humanToll protein, designated PRO286 (SEQ ID NO: 3).

FIG. 4 shows the nucleotide sequence of a native sequence human Tollprotein cDNA designated DNA42663 (SEQ ID NO: 4), which encodes PRO286.

FIG. 5 shows the expression pattern of human Toll receptor 2 (huTLR2)(Rock et al., supra). a. Northern analysis of human multiple immunetissues probed with a TLR2 probe. PBL, peripheral blood leukocytes. b.Enriched expression of TLR2 in macrophages, and transcriptionalup-regulation of TLR2 in response to LPS. Quantitative RT-PCR was usedto determined the relative expression levels of TLR2 in PBL, T cells,macrophages (MΦ), and LPS-stimulated macrophages (MΦ+LPS).

FIG. 6 TLR2 mediates LPS-induced signaling. a. 293 cells stablyexpressing TLR2 acquire LPS responsiveness. Either a population ofstable clones expressing gD.TLR2 (293-TLR2 pop1) or a single clone ofcells expressing gD.TLR2 (293-TLR2 clone 1) or control cells (293-MSCV)that were stably transfected with the expression vector alone weretransiently transfected with pGL3.ELAM.tk and then stimulated with 1μg/ml of 055:B5 enhancer for 6 h with or without LBP in serum-freemedium. Activation of the ELAM enhancer was measured as described in theExamples. Results were obtained from two independent experiments. Nostimulation was observed using the control reporter plasmid that lackedthe ELAM enhancer (data not sown). Expression of the reporter plasmidwas equivalent in untreated cells or cells treated with LBP alone (datanot shown). b. Western blot showing expression of epitope-tagged TLR2 in293 cells. c. Time course of TLR2-dependent LPS-induced activation andtranslocation of NF-κB. Nuclear extracts were prepared from cellstreated with 055:B5 LPS (10 μg/ml) and LBP for the indicated times(top), or cells pretreated with 1 μM cycloheximide (CHX) for 1 h thestimulated with 1 μg/ml LPS for 1 h in the presence of LBP in serum-freemedium (bottom). d. Effect of mCD14 on NF-κB activation by TLR2. Vectorcontrol (193-MSCV) or 293-TLR2 pop1 cells were transfected with thereporter plasmid, and a CD14 expression vector (+mCD14) or vectorcontrol (−mCD14), respectively. After 24 h, transfected cells werestimulated with 055:B5 LPS for 6 h in the presence of LBP in serum-freemedium. The data presented are representative from three independentexperiments.

FIG. 7 Domain function of TLR2 in signaling. a. Illustrations of variousTLR2 constructs. TLR2-WT, the full-length epitope-tagged form of TLR2,TLR2-Δ1 and -Δ2 represent a truncation of 13 or 141 amino acids at thecarboxyl terminus, respectively. CD4-TLR2, a human CD4-TLR2 chimerareplacing the extracellular domain of TLR2 with amino acids 1-205 ofhuman CD4. ECD, extracellular domain; TM, transmembrane region; ICD,intracellular domain. b. C-terminal residues critical for IL-1R and TLR2signal transduction. Residue numbers are shown to the right of eachprotein. Arrow indicated the position of the TLR2-Δ1 truncation. *,residues essential for IL-1R signaling (Heguy et al., J. Biol. Chem.267, 2605-2609 [1992]; Croston et al., J. Biol. Chem. 27o, 16514-16517[1995])l I, identical amino acid; conservative changes. c. TLR-R2variants fail to induce NF-κB in response to LPS and LBP. 293 cells weretransiently transfected with pGL3.ELAM.tk and expression vectorsencoding full-length TLR2 or TLR2 variants as indicated. The cells werealso transfected with a CD14 expression plasmid (+mCD14) or with acontrol plasmid (−mCD14). Equal expression of each protein is confirmedby Western blot using either anti-gD or CD4 antibody (FIG. 7D). Theluciferase assay was performed as described in the Examples. Data wereobtained from duplicate experiments.

FIG. 8 High potency of E. coli K12 LPS (LCD25) and its binding to TLR2.a. Dose-response curve of various LPS preparations. b. Specificinteraction of [³H]-LPS (LCD25) with the extracellular domain of TLR2.Specific binding was observed to TLR2-Fc, but not to either Fc alone, orfusion proteins containing the extracellular domains of Rse, Axl, Her2,or Her4. Binding to TLR2-Fc was specifically competed with LCD25 LPS,but not with detoxified LPS.

FIG. 9 TLR2 is required for the LPS-induced IL-8 expression. 293-MSCVvector control and 293-TLR2 cells transiently expressing mCD14 werestimulated with LBP alone or together with the indicated type of LPS atconcentrations of 1 μg/ml in serum-free medium for 6 h. Equal amounts ofpoly-(A) RNAs were used for Northern analysis.

FIG. 10 Nucleotide sequence encoding huTLR2 (SEQ ID NO:11).

FIG. 11 Amino acid sequence of huTLR2 (SEQ ID NO:12).

FIGS. 12A-C show the derived amino acid sequence of a native sequencehuman Toll protein, designated PRO358 (SEQ ID NO: 13). In the Figure,amino acids 1 through 19 form a putative signal sequence, amino acids 20through 575 are the putative extracellular domain, with amino acids 20through 54 having the characteristics of leucine rich repeats, aminoacids 576 through 595 are a putative transmembrane domain, whereas aminoacids 596 through 811 form an intracellular domain.

FIGS. 13A-B (SEQ ID NO: 14) show the nucleotide sequence of a nativesequence human Toll protein cDNA designated DNA47361, which encodes themature, full-length Toll protein, PRO358. As the sequence shown containssome extraneous sequences, the ATG start codon is underlined, and theTAA stop codon is boxed.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions

The terms “PRO285 polypeptide”, “PRO286 polypeptide”, “PRO285” and“PRO286”, when used herein, encompass the native sequence PRO285 andPRO286 Toll proteins and variants (which are further defined herein).The PRO285 and PRO286 polypeptide may be isolated from a variety ofsources, such as from human tissue types or from another source, orprepared by recombinant or synthetic methods, or by any combination ofthese and similar techniques.

A “native sequence PRO285” or “native sequence PRO286” comprises apolypeptide having the same amino acid sequence as PRO285 or PRO286derived from nature. Such native sequence Toll polypeptides can beisolated from nature or can be produced by recombinant or syntheticmeans. The terms “native sequence PRO285” and “native sequence PRO286”specifically encompass naturally-occurring truncated or secreted formsof the PRO285 and PRO286 polypeptides disclosed herein (e.g., anextracellular domain sequence), naturally-occurring variant forms (e.g.,alternatively spliced forms) and naturally-occurring allelic variants ofthe PRO285 and PRO286 polypeptides. In one embodiment of the invention,the native sequence PRO285 is a mature or full-length native sequencePRO285 polypeptide comprising amino acids 1 to 1049 of FIG. 1 (SEQ IDNO: 1), while native sequence PRO286 is a mature or full-length nativesequence PRO286 polypeptide comprising amino acids 1 to 1041 of FIG. 3(SEQ ID NO:3). In a further embodiment, the native sequence PRO285comprises amino acids 27-1049, or 27-836 of FIG. 1 (SEQ ID NO:1), oramino acids 27-1041, or 27-825 of FIG. 3 (SEQ ID NO:3).

The terms “PRO285 variant” and “PRO286 variant” mean an active PRO285 orPRO286 polypeptide as defined below having at least about 80% amino acidsequence identity with PRO285 having the deduced amino acid sequenceshown in FIG. 1 (SEQ ID NO:1) for a full-length native sequence PRO285,or at least about 80% amino acid sequence identity with PRO286 havingthe deduced amino acid sequence shown in FIG. 3 (SEQ ID NO:3) for afull-length native sequence PRO286. Such variants include, for instance,PRO285 and PRO286 polypeptides wherein one or more amino acid residuesare added, or deleted, at the N- or C-terminus of the sequences of FIGS.1 and 3 (SEQ ID NO: 1 and 3), respectively. Ordinarily, a PRO285 orPRO286 variant will have at least about 80% amino acid sequenceidentity, more preferably at least about 90% amino acid sequenceidentity, and even more preferably at least about 95% amino acidsequence identity with the amino acid sequence of FIG. 1 or FIG. 3 (SEQID NOs:1 and 3). Preferred variants are those which show a high degreeof sequence identity with the extracellular domain of a native sequencePRO285 or PRO286 polypeptide. In a special embodiment, the PRO285 andPRO286 variants of the present invention retain at least a C-terminalportion of the intracellular domain of the corresponding nativeproteins, and most preferably they retain most of the intracellular andthe extracellular domains. However, depending on their intended use,such variants may have various amino acid alterations, e.g.,substitutions, deletions and/or insertions within these regions.

The terms “PRO358 polypeptide”, “PRO358”, “PRO358 Toll homologue” andgrammatical variants thereof, as used herein, encompass the nativesequence PRO358 Toll protein and variants (which are further definedherein). The PRO358 polypeptide may be isolated from a variety ofsources, such as from human tissue types or from another source, orprepared by recombinant or synthetic methods, or by any combination ofthese and similar techniques.

A “native sequence PRO358” comprises a polypeptide having the same aminoacid sequence as PRO358 derived from nature. Such native sequence Tollpolypeptides can be isolated from nature or can be produced byrecombinant or synthetic means. The term “native sequence PRO358”specifically encompasses naturally-occurring truncated or secreted formsof the PRO358 polypeptide disclosed herein (e.g., an extracellulardomain sequence), naturally-occurring variant forms (e.g., alternativelyspliced forms) and naturally-occurring allelic variants. In oneembodiment of the invention, the native sequence PRO358 is a mature orfull-length native sequence PRO358 polypeptide comprising amino acids 20to 811 of FIGS. 12A-C (SEQ ID NO: 13), with or without the N-terminalsignal sequence (amino acids 1 to 19), and with or without theN-terminal methionine. In another embodiment, the native sequence PRO358is the soluble form of the full-length PRO358, retaining theextracellular domain of the full-length protein (amino acids 29 to 575),with or without the N-terminal signal sequence, and with or without theN-terminal methionine.

The term “PRO358 variant” means an active PRO358 polypeptide as definedbelow having at least about 80%, preferably at least about 85%, morepreferably at least about 90%, most preferably at least about 95% aminoacid sequence identity with PRO358 having the deduced amino acidsequence shown in FIGS. 12A-C (SEQ ID NO:13). Such variants include, forinstance, PRO358 polypeptides wherein one or more amino acid residuesare added, or deleted, at the N- or C-terminus of the sequences of FIGS.12A-C (SEQ ID NO:13). Variants specifically include transmembrane-domaindeleted and inactivated variants of native sequence PRO358, which mayalso have part or whole of their intracellular domain deleted. Preferredvariants are those which show a high degree of sequence identity withthe extracellular domain of the native sequence PRO358 polypeptide. In aspecial embodiment, the PRO358 variants of the present invention retainat least a C-terminal portion of the intracellular domain of acorresponding native protein, and most preferably they retain most ofthe intracellular and the extracellular domains. However, depending ontheir intended use, such variants may have various amino acidalterations, e.g., substitutions, deletions and/or insertions withinthese regions.

“Percent (%) amino acid sequence identity” with respect to the PRO285,PRO286 and PRO358 sequences identified herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in the PRO285, PRO286, or PRO358sequence, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Alignment for purposes of determining percent amino acidsequence identity can be achieved in various ways that are within theskill in the art, for instance, using publicly available computersoftware such as BLAST, ALIGN or Megalign (DNASTAR) software. Thoseskilled in the art can determine appropriate parameters for measuringalignment, including any algorithms needed to achieve maximal alignmentover the full length of the sequences being compared. The ALIGN softwareis preferred to determine amino acid sequence identity.

In a specific aspect, “percent (%) amino acid sequence identity” withrespect to the PRO285, PRO286 and PRO358 sequences identified herein isdefined as the percentage of amino acid residues in a candidate sequencethat are identical with the amino acid residues in the PRO285, PRO286and PRO358 sequence, after aligning the sequences and introducing gaps,if necessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. The % identity values used herein are generated by WU-BLAST-2which was obtained from [Altschul et al., Methods in Enzymology, 266:460-480 (1996); http://blast.wustl/edu/blast/README.html]. WU-BLAST-2uses several search parameters, most of which are set to the defaultvalues. The adjustable parameters are set with the following values:overlap span=1, overlap fraction=0.125, word threshold (T)=11. The HSP Sand HSP S2 parameters are dynamic values and are established by theprogram itself depending upon the composition of the particular sequenceand composition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity. A % amino acid sequence identity value isdetermined by the number of matching identical residues divided by thetotal number of residues of the “longer” sequence in the aligned region.The “longer” sequence is the one having the most actual residues in thealigned region (gaps introduced by WU-Blast-2 to maximize the alignmentscore are ignored).

The term “positives”, in the context of sequence comparison performed asdescribed above, includes residues in the sequences compared that arenot identical but have similar properties (e.g. as a result ofconservative substitutions). The % value of positives is determined bythe fraction of residues scoring a positive value in the BLOSUM 62matrix divided by the total number of residues in the longer sequence,as defined above.

“Percent (%) nucleic acid sequence identity” with respect to theDNA40021, DNA42663 and DNA47361 sequences identified herein is definedas the percentage of nucleotides in a candidate sequence that areidentical with the nucleotides in the DNA40021, DNA42663 and DNA47361sequences, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. Alignmentfor purposes of determining percent nucleic acid sequence identity canbe achieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,ALIGN or Megalign (DNASTAR) software. Those skilled in the art candetermine appropriate parameters for measuring alignment, including anyalgorithms needed to achieve maximal alignment over the full length ofthe sequences being compared. The ALIGN software is preferred todetermine nucleic acid sequence identity.

Specifically, “percent (%) nucleic acid sequence identity” with respectto the coding sequence of the PRO285, PRO286 and PRO358 polypeptidesidentified herein is defined as the percentage of nucleotide residues ina candidate sequence that are identical with the nucleotide residues inthe PRO285, PRO286 and PRO358 coding sequence. The identity values usedherein were generated by the BLASTN module of WU-BLAST-2 set to thedefault parameters, with overlap span and overlap fraction set to 1 and0.125, respectively.

“Isolated,” when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the PRO285, PRO286,or PRO358 natural environment will not be present. Ordinarily, however,isolated polypeptide will be prepared by at least one purification step.

An “isolated” DNA40021, DNA42663 or DNA47361 nucleic acid molecule is anucleic acid molecule that is identified and separated from at least onecontaminant nucleic acid molecule with which it is ordinarily associatedin the natural source of the DNA40021, DNA42663 or DNA47361 nucleicacid. An isolated DNA40021, DNA42663 or DNA47361 nucleic acid moleculeis other than in the form or setting in which it is found in nature.Isolated DNA40021, DNA42663 and DNA47361 nucleic acid moleculestherefore are distinguished from the DNA40021, DNA42663 or DNA47361nucleic acid molecule as it exists in natural cells. However, anisolated DNA40021, DNA42663 or DNA47361 nucleic acid molecule includesDNA40021, DNA42663 and DNA47361 nucleic acid molecules contained incells that ordinarily express DNA40021, DNA42663 or DNA47361 where, forexample, the nucleic acid molecule is in a chromosomal locationdifferent from that of natural cells.

“Toll receptor2”, “TLR2” and “huTLR2” are used interchangeably, andrefer to a human Toll receptor designated as “HuTLR2” by Rock et al.,Proc. Natl. Acad. Sci. USA 95, 588-593 (1998). The nucleotide and aminoacid sequences of huTLR2 are shown in FIGS. 10 (SEQ ID NO: 11) and 11(SEQ ID NO: 12), respectively.

The term “expression vector” is used to define a vector, in which anucleic acid encoding a Toll homologue protein herein is operably linkedto control sequences capable of affecting its expression is a suitablehost cells. Vectors ordinarily carry a replication site (although thisis not necessary where chromosomal integration will occur). Expressionvectors also include marker sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies (Bolivar, et al., Gene 2: 95 [1977]). pBR322 contains genes forampicillin and tetracycline resistance and thus provides easy means foridentifying transformed cells, whether for purposes of cloning orexpression. Expression vectors also optimally will contain sequenceswhich are useful for the control of transcription and translation, e.g.,promoters and Shine-Dalgarno sequences (for prokaryotes) or promotersand enhancers (for mammalian cells). The promoters may be, but need notbe, inducible; even powerful constitutive promoters such as the CMVpromoter for mammalian hosts have been found to produce the LHR withouthost cell toxicity. While it is conceivable that expression vectors neednot contain any expression control, replicative sequences or selectiongenes, their absence may hamper the identification of hybridtransformants and the achievement of high level hybrid immunoglobulinexpression.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The term “antibody” is used in the broadest sense and specificallycovers single anti-PRO285, anti-PRO286 and anti-PRO358 monoclonalantibodies (including agonist, antagonist, and neutralizing antibodies)and anti-PRO285, anti-PRO286 and anti-PRO358 antibody compositions withpolyepitopic specificity. The term “monoclonal antibody” as used hereinrefers to an antibody obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally-occurringmutations that may be present in minor amounts.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, prevents, inhibits, orneutralizes a biological activity of a native Toll receptor disclosedherein. In a similar manner, the term “agonist” is used in the broadestsense and includes any molecule that mimics, or enhances a biologicalactivity of a native Toll receptor disclosed herein. Suitable agonist orantagonist molecules specifically include agonist or antagonistantibodies or antibody fragments, fragments or amino acid sequencevariants of native Toll receptor polypeptides, peptides, small organicmolecules, etc.

“Active” or “activity” for the purposes herein refers to form(s) ofPRO285, PRO286 and PRO358 which retain the biologic and/or immunologicactivities of native or naturally-occurring PRO285, PRO286 and PRO358,respectively. A preferred “activity” is the ability to induce theactivation of NF-κB and/or the expression of NF-κB-controlled genes forthe inflammatory cytokines IL-1, IL-6 and IL-8. Another preferred“activity” is the ability to activate an innate and/or adaptive immuneresponse in vertebrates. A further preferred “activity” is the abilityto sense the presence of conserved molecular structures present onmicrobes, and specifically the ability to mediate lipopolysaccharide(LPS) signaling. The same “activity” definition applies to agonists(e.g. agonist antibodies) of PRO285, PRO286 and PRO358 polypeptides. Asnoted above, the “activity” an antagonist (including agonist antibodies)of a PRO285, PRO286 or PRO358 polypeptide is defined as the ability tocounteract, e.g. partially or fully block, prevent, inhibit, orneutralize any of the above-identified activities of a PRO285, PRO286 orPRO358 polypeptide.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; (3) employ50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodiumcitrate) and 50% formamide at 55° C., followed by a high-stringency washconsisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising a FIZZ polypeptide fused to a “tag polypeptide.”The tag polypeptide has enough residues to provide an epitope againstwhich an antibody can be made, yet is short enough such that it does notinterfere with activity of the polypeptide to which it is fused. The tagpolypeptide preferably also is fairly unique so that the antibody doesnot substantially cor-cross-react with other epitopes. Suitable tagpolypeptides generally have at least six amino acid residues and usuallybetween about 8 and 50 amino acid residues (preferably, between about 10and 20 amino acid residues).

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesin comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and the immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder. Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, cats, cows, horses, sheep, pigs, etc.Preferably, the mammal is human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

The term “lipopolysaccharide” or “LPS” is used herein as a synonym of“endotoxin.” Lipopolysaccharides (LPS) are characteristic components ofthe outer membrane of Gram-negative bacteria, e.g., Escherichia coli.They consist of a polysaccharide part and a fat called lipid A. Thepolysaccharide, which varies from one bacterial species to another, ismade up of the O-specific chain (built from repeating units of three toeight sugars) and the two-part core. Lipid A virtually always includestwo glucosamine sugars modified by phosphate and a variable number offatty acids. For further information see, for example, Rietschel andBrade, Scientific American August 1992, 54-61.

The term “septic shock” is used herein in the broadest sense, includingall definitions disclosed in Bone, Ann. Intern Med. 114, 332-333 (1991).Specifically, septic shock starts with a systemic response to infection,a syndrome called sepsis. When this syndrome results in hypotension andorgan dysfunction, it is called septic shock. Septic shock may beinitiated by gram-positive organisms and fungi, as well asendotoxin-containing Gram-negative organisms. Accordingly, the presentdefinition is not limited to “endotoxin shock.”

II. Compositions and Methods of the Invention

A. Full-Length PRO285, PRO286 and PRO358

The present invention provides newly identified and isolated nucleotidesequences encoding, polypeptides referred to in the present applicationas PRO285 and PRO286 In particular, Applicants have identified andisolated cDNAs encoding PRO285 and PRO286 polypeptides, as disclosed infurther detail in the Examples below. Using BLAST and FastA sequencealignment computer programs, Applicants found that the coding sequencesof PRO285 and PRO286 are highly homologous to DNA sequencesHSU88540_(—)1, HSU88878_(—)1, HSU88879_(—)1, HSU88880_(—)1, andHSU88881_(—)1 in the GenBank database.

The present invention further provides newly identified and isolatednucleotide sequences encoding a polypeptide referred to in the presentapplication as PRO358. In particular, Applicants have identified andisolated cDNA encoding a novel human Toll polypeptide (PRO358), asdisclosed in further detail in the Examples below. Using BLAST and FastAsequence alignment computer programs, Applicants found that the codingsequence of PRO358 shows significant homology to DNA sequencesHSU88540_(—)1, HSU88878_(—)1, HSU88879_(—)1, HSU88880_(—)1,HS88881_(—)1, and HSU79260_(—)1 in the GenBank database. With theexception of HSU79260_(—)1, the noted proteins have been identified ashuman toll-like receptors.

Accordingly, it is presently believed that the PRO285, PRO286 and PRO358proteins disclosed in the present application are newly identified humanhomologues of the Drosophila protein Toll, and are likely to play animportant role in adaptive immunity. More specifically, PRO285, PRO286and PRO358 may be involved in inflammation, septic shock, and responseto pathogens, and play possible roles in diverse medical conditions thatare aggravated by immune response, such as, for example, diabetes, ALS,cancer, rheumatoid arthritis, and ulcers. The role of PRO285, PRO286 andPRO385 as pathogen pattern recognition receptors, sensing the presenceof conserved molecular structures present on microbes, is furthersupported by the data disclosed in the present application, showing thata known human Toll-like receptor, TLR2 is a direct mediator of LPSsignaling.

B. PRO285. PRO286 and PRO358 Variants

In addition to the full-length native sequence PRO285, PRO286 and PRO358described herein, it is contemplated that variants of these sequencescan be prepared. PRO285, PRO286 and PRO358 variants can be prepared byintroducing appropriate nucleotide changes into the PRO285, PRO286 orPRO358 DNA, or by synthesis of the desired variant polypeptides. Thoseskilled in the art will appreciate that amino acid changes may alterpost-translational processes of the PRO285, PRO286 or PRO358polypeptides, such as changing the number or position of glycosylationsites or altering the membrane anchoring characteristics.

Variations in the native full-length sequence PRO285, PRO286 or PRO358,or in various domains of the PRO285, PRO286, or PRO358 described herein,can be made, for example, using any of the techniques and guidelines forconservative and non-conservative mutations set forth, for instance, inU.S. Pat. No. 5,364,934. Variations may be a substitution, deletion orinsertion of one or more codons encoding the PRO285, PRO286, or PRO358polypeptide that results in a change in the amino acid sequence ascompared with the corresponding native sequence polypeptides. Optionallythe variation is by substitution of at least one amino acid with anyother amino acid in one or more of the domains of the PRO285, PRO286, orPRO358. Guidance in determining which amino acid residue may beinserted, substituted or deleted without adversely affecting the desiredactivity may be found by comparing the sequence of the PRO285, PRO286,or PRO358 with that of homologous known protein molecules and minimizingthe number of amino acid sequence changes made in regions of highhomology. Amino acid substitutions can be the result of replacing oneamino acid with another amino acid having similar structural and/orchemical properties, such as the replacement of a leucine with a serine,i.e., conservative amino acid replacements. Insertions or deletions mayoptionally be in the range of 1 to 5 amino acids. The variation allowedmay be determined by systematically making insertions, deletions orsubstitutions of amino acids in the sequence and testing the resultingvariants for activity in the in vitro assay described in the Examplesbelow.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the PRO285 or PRO286 variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant. Alanine is alsotypically preferred because it is the most common amino acid. Further,it is frequently found in both buried and exposed positions [Creighton,The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1(1976)]. If alanine substitution does not yield adequate amounts ofvariant, an isoteric amino acid can be used.

Variants of the PRO285, PRO286 and PRO358 Toll proteins disclosed hereininclude proteins in which the transmembrane domains have been deleted orinactivated. Transmembrane regions are highly hydrophobic or lipophilicdomains that are the proper size to span the lipid bilayer of thecellular membrane. They are believed to anchor the native, maturePRO285, PRO286 and PRO358 polypeptides in the cell membrane. In PRO285the transmembrane domain stretches from about amino acid position 840 toabout amino acid position 864. In PRO286 the transmembrane domain isbetween about amino acid position 826 and about amino acid position 848.In PRO358 the transmembrane domain is between about amino acid position576 and amino acid position 595.

Deletion or substitution of the transmembrane domain will facilitaterecovery and provide a soluble form of a PRO285, PRO286, and PRO358polypeptide by reducing its cellular or membrane lipid affinity andimproving its water solubility. If the transmembrane and cytoplasmicdomains are deleted one avoids the introduction of potentiallyimmunogenic epitopes, either by exposure of otherwise intracellularpolypeptides that might be recognized by the body as foreign or byinsertion of heterologous polypeptides that are potentially immunogenic.A principal advantage of a transmembrane domain deleted PRO285, PRO286or PRO358 is that it is secreted into the culture medium of recombinanthosts. This variant is soluble in body fluids such as blood and does nothave an appreciable affinity for cell membrane lipids, thus considerablysimplifying its recovery from recombinant cell culture.

It will be amply apparent from the foregoing discussion thatsubstitutions, deletions, insertions or any combination thereof areintroduced to arrive at a final construct. As a general proposition,soluble variants will not have a functional transmembrane domain andpreferably will not have a functional cytoplasmic sequence. This isgenerally accomplished by deletion of the relevant domain, althoughadequate insertional or substitutional variants also are effective forthis purpose. For example, the transmembrane domain is substituted byany amino acid sequence, e.g. a random or predetermined sequence ofabout 5 to 50 serine, threonine, lysine, arginine, glutamine, asparticacid and like hydrophilic residues, which altogether exhibit ahydrophilic hydropathy profile. Like the deletional (truncated) PRO285,PRO286 and PRO358 variants, these variants are secreted into the culturemedium of recombinant hosts.

Further deletional variants of the full-length mature PRO285, PRO286,and PRO358 polypeptides (or transmembrane domain deleted to inactivatedforms thereof) include variants from which the N-terminal signal peptide(putatively identified as amino acids 1 to 19 for PRO285 and PRO286, andas amino acids 1 to 26 for PRO358) and/or the initiating methionine hasbeen deleted. The native signal sequence may also be substituted byanother (heterologous) signal peptide, which may be that of anotherToll-like protein, or another human or non-human (e.g., bacterial, yeastor non-human mammalian) signal sequence.

It is believed that the intracellular domain, and especially itsC-terminal portion, is important for the biological function of thesepolypeptides. Accordingly, if the objective is to make variants whichretain the biological activity of a corresponding native Toll-likeprotein, at least a substantial portion of these regions is retain, orthe alterations, if any, involve conservative amino acid substitutionsand/or insertions or amino acids which are similar in character to thosepresent in the region where the amino acid is inserted. If, however, asubstantial modification of the biological function of a native Tollreceptor is required (e.g., the objective is to prepare antagonists ofthe respective native Toll polypeptides), the alterations involve thesubstitution and/or insertion of amino acids, which differ in characterfrom the amino acid at the targeted position in the corresponding nativeToll polypeptide.

Naturally-occurring amino acids are divided into groups based on commonside chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophobic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Conservative substitutions involve exchanging a member within one groupfor another member within the same group, whereas non-conservativesubstitutions will entail exchanging a member of one of these classesfor another. Variants obtained by non-conservative substitutions areexpected to result in more significant changes in the biologicalproperties/function of the obtained variant.

Amino acid insertions include amino- and/or carboxyl-terminal fusionsranging in length from one residue to polypeptides containing a hundredor more residues, as well as intrasequence insertions of single ormultiple amino acid residues. Intrasequence insertions (i.e. insertionswithin the PRO285, PRO286 or PRO358 protein amino acid sequence) mayrange generally from about 1 to 10 residues, more preferably 1 to 5residues, more preferably 1 to 3 residues. Examples of terminalinsertions include the PRO285, PRO286 and PRO358 polypeptides with anN-terminal methionyl residue, an artifact of its direct expression inbacterial recombinant cell culture, and fusion of a heterologousN-terminal signal sequence to the N-terminus of the PRO285, PRO286, orPRO358 molecule to facilitate the secretion of the mature I-TRAFproteins from recombinant host cells. Such signal sequences willgenerally be obtained from, and thus homologous to, the intended hostcell species. Suitable sequences include STII or Ipp for E. coli, alphafactor for yeast, and viral signals such as herpes gD for mammaliancells.

Other insertional variants of the native Toll-like molecules disclosedherein include the fusion of the N- or C-terminus of the native sequencemolecule to immunogenic polypeptides, e.g. bacterial polypeptides suchas beta-lactamase or an enzyme encoded by the E. coli trp locus, oryeast protein, and C-terminal fusions with proteins having a longhalf-life such as immunoglobulin regions (preferably immunoglobulinconstant regions to yield immunoadhesins), albumin, or ferritin, asdescribed in WO 89/02922 published on 6 Apr. 1989. For the production ofimmunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27,1995.

Since it is often difficult to predict in advance the characteristics ofa variant Toll-like protein, it will be appreciated that screening willbe needed to select the optimum variant. For this purpose biochemical orother screening assays, such as those described hereinbelow, will bereadily available.

C. Modifications of the PRO285, PRO286 and PRO358 Toll Proteins

Covalent modifications of the PRO285, PRO286 and PRO358 human Tollhomologues are included within the scope of this invention. One type ofcovalent modification includes reacting targeted amino acid residues ofthe PRO285, PRO286 or PRO358 protein with an organic derivatizing agentthat is capable of reacting with selected side chains or the N- orC-terminal residues. Derivatization with bifunctional agents is useful,for instance, for crosslinking PRO285, PRO286, or PRO358 to awater-insoluble support matrix or surface for use in the method forpurifying anti-PRO285-PRO286, or -PRO358 antibodies, and vice-versa.Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis-(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Derivatization with bifunctional agents is useful for preparingintramolecular aggregates of the Toll-like receptors herein withpolypeptides as well as for cross-linking these polypeptides to a waterinsoluble support matrix or surface for use in assays or affinitypurification. In addition, a study of interchain cross-links willprovide direct information on conformational structure. Commonly usedcross-linking agents include 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, homobifunctionalimidoesters, and bifunctional maleimides. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates which are capable of forming cross-links in the presenceof light. Alternatively, reactive water insoluble matrices such ascyanogen bromide activated carbohydrates and the systems reactivesubstrates described in U.S. Pat. Nos. 3,959,642; 3,969,287; 3,691,016;4,195,128; 4,247,642; 4,229,537; 4,055,635; and 4,330,440 are employedfor protein immobilization and cross-linking.

Another type of covalent modification of the PRO285, PRO286 and PRO358polypeptides included within the scope of this invention comprisesaltering the native glycosylation pattern of the polypeptide. “Alteringthe native glycosylation pattern” is intended for purposes herein tomean deleting one or more carbohydrate moieties found in native sequence(either by removing the underlying glycosylation site or by deleting theglycosylation by chemical and/or enzymatic means) and/or adding one ormore glycosylation sites that are not present in the native sequence. Inaddition, the phrase includes qualitative changes in the glycosylationof the native proteins, involving a change in the nature and proportionsof the carbohydrates present.

The native, full-length PRO285 (encoded by DNA 40021) has potentialN-linked glycosylation sites at the following amino acid positions: 66,69, 167, 202, 215, 361, 413, 488, 523, 534, 590, 679, 720, 799 and 942.The native, full-length PRO286 (encoded by DNA42663) has potentialN-linked glycosylation sites at the following amino acid positions: 29,42, 80, 88, 115, 160, 247, 285, 293, 358, 362, 395, 416, 443, 511, 546,582, 590, 640, 680, 752, 937 and 1026.

Addition of glycosylation sites to the PRO285, PRO286 and PRO358polypeptides may be accomplished by altering the amino acid sequence.The alteration may be made, for example, by the addition of, orsubstitution by, one or more serine or threonine residues to the nativesequence (for O-linked glycosylation sites). The amino acid sequence mayoptionally be altered through changes at the DNA level, particularly bymutating the DNA encoding the PRO285, PRO286, and PRO358 polypeptides atpreselected bases such that codons are generated that will translateinto the desired amino acids.

Another means of increasing the number of carbohydrate moieties on thePRO285, PRO286 and PRO358 polypeptides is by chemical or enzymaticcoupling of glycosides to the polypeptide. Such methods are described inthe art, e.g., in WO 87/05330 published 11 Sep. 1987, and in Aplin andWriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the PRO285, PRO286 andPRO358 polypeptides may be accomplished chemically or enzymatically orby mutational substitution of codons encoding for amino acid residuesthat serve as targets for glycosylation. Chemical deglycosylationtechniques are known in the art and described, for instance, byHakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987) and by Edgeet al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage ofcarbohydrate moieties on polypeptides can be achieved by the use of avariety of endo- and exo-glycosidases as described by Thotakura et al.,Meth. Enzymol., 138:350 (1987).

Another type of covalent modification comprises linking the PRO285,PRO286 and PRO358 polypeptides to one of a variety of nonproteinaceouspolymers, e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. No. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The PRO285, PRO286 and PRO358 polypeptides of the present invention mayalso be modified in a way to form a chimeric molecule comprising PRO285,PRO286, PRO358, or a fragment thereof, fused to another, heterologouspolypeptide or amino acid sequence. In one embodiment, such a chimericmolecule comprises a fusion of the PRO285, PRO286 or PRO358 polypeptidewith a tag polypeptide which provides an epitope to which an anti-tagantibody can selectively bind. The epitope tag is generally placed atthe amino- or carboxyl-terminus of a native or variant PRO285, PRO286,or PRO358 molecule. The presence of such epitope-tagged forms can bedetected using an antibody against the tag polypeptide. Also, provisionof the epitope tag enables the PRO285, PRO286, or PRO358 polypeptides tobe readily purified by affinity purification using an anti-tag antibodyor another type of affinity matrix that binds to the epitope tag.

Various tag polypeptides and their respective antibodies are well knownin the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 3:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; an α-tubulin epitope peptide [Skinneret al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)].

In a further embodiment, the chimeric molecule may comprise a fusion ofthe PRO285, PRO286 or PRO358 polypeptides, or fragments thereof, with animmunoglobulin or a particular region of an immunoglobulin. For abivalent form of the chimeric molecule, such a fusion could be to the Fcregion of an Ig, such as, IgG molecule. The Ig fusions preferablyinclude the substitution of a soluble (transmembrane domain deleted orinactivated) form of a PRO285, PRO286, or PRO358 polypeptide in place ofat least one variable region within an Ig molecule. For the productionof immunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued Jun.27, 1995.

D. Preparation of PRO285, PRO286 and PRO358 Polypeptides

The description below relates primarily to production of PRO285, PRO286,and PRO358 Toll homologues by culturing cells transformed or transfectedwith a vector containing nucleic acid encoding these proteins (e.g.DNA40021, DNA42663, and DNA47361, respectively). It is, of course,contemplated that alternative methods, which are well known in the art,may be employed to prepare PRO285, PRO286, PRO358, or their variants.For instance, the PRO285, PRO286 or PRO358 sequence, or portionsthereof, may be produced by direct peptide synthesis using solid-phasetechniques [see, e.g., Stewart et al., Solid-Phase Peptide Synthesis,W.H. Freeman Co., San Francisco, Calif. (1969); Merrifield, J. Am. Chem.Soc., 85:2149-2154 (1963)]. In vitro protein synthesis may be performedusing manual techniques or by automation. Automated synthesis may beaccomplished, for instance, using an Applied Biosystems PeptideSynthesizer (Foster City, Calif.) using manufacturer's instructions.Various portions of the PRO285, PRO286, or PRO358 may be chemicallysynthesized separately and combined using chemical or enzymatic methodsto produce the full-length PRO285, PRO286, or PRO358.

1. Isolation of DNA Encoding PRO285 PRO286 or PRO358

DNA encoding PRO285, PRO286, or PRO358 may be obtained from a cDNAlibrary prepared from tissue believed to possess the PRO285, PRO286, orPRO358 mRNA and to express it at a detectable level. Accordingly, humanPRO285, PRO286, or PRO358 DNA can be conveniently obtained from a cDNAlibrary prepared from human tissue, such as described in the Examples.The underlying gene may also be obtained from a genomic library or byoligonucleotide synthesis. In addition to the libraries described in theExamples, DNA encoding the human Toll proteins of the present inventioncan be isolated, for example, from spleen cells, or peripheral bloodleukocytes (PBL).

Libraries can be screened with probes (such as antibodies to the PRO285,PRO286, or PRO358 protein or oligonucleotides of at least about 20-80bases) designed to identify the gene of interest or the protein encodedby it. Screening the cDNA or genomic library with the selected probe maybe conducted using standard procedures, such as described in Sambrook etal., Molecular Cloning: A Laboratory Manual (New York: Cold SpringHarbor Laboratory Press, 1989). An alternative means to isolate the geneencoding PRO285, PRO286, or PRO358 is to use PCR methodology [Sambrooket al., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (ColdSpring Harbor Laboratory Press, 1995)].

The Examples below describe techniques for screening a cDNA library. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra.

Sequences identified in such library screening methods can be comparedand aligned to other known sequences deposited and available in publicdatabases such as GenBank or other private sequence databases. Sequenceidentity (at either the amino acid or nucleotide level) within definedregions of the molecule or across the full-length sequence can bedetermined through sequence alignment using computer software programssuch as ALIGN, DNAstar, and INHERIT which employ various algorithms tomeasure homology/sequence identity.

Nucleic acid having protein coding sequence may be obtained by screeningselected cDNA or genomic libraries using the deduced amino acid sequencedisclosed herein for the first time, and, if necessary, usingconventional primer extension procedures as described in Sambrook etal., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

2. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for the production of the human Toll proteinsand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences. The culture conditions, such as media,temperature, pH and the like, can be selected by the skilled artisanwithout undue experimentation. In general, principles, protocols, andpractical techniques for maximizing the productivity of cell culturescan be found in Mammalian Cell Biotechnology: a Practical Approach, M.Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.

Methods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ and electroporation. Depending on the host cell used,transformation is performed using standard techniques appropriate tosuch cells. The calcium treatment employing calcium chloride, asdescribed in Sambrook et al., supra, or electroporation is generallyused for prokaryotes or other cells that contain substantial cell-wallbarriers. Infection with Agrobacterium tumefaciens is used fortransformation of certain plant cells, as described by Shaw et al.,Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 2:456-457(1978) can be employed. General aspects of mammalian cell host systemtransformations have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as K coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635).

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for humanToll-encoding vectors. Saccharomyces cerevisiae is a commonly used lowereukaryotic host microorganism

Suitable host cells for the expression of glycosylated human Tollproteins are derived from multicellular organisms. Examples ofinvertebrate cells include insect cells such as Drosophila S2 andSpodoptera Sf9, as well as plant cells. Examples of useful mammalianhost cell lines include Chinese hamster ovary (CHO) and COS cells. Morespecific examples include monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. GenVirol., 36:59 (1977)); Chinese hamster ovary cells/−DHFR (CHO, Urlauband Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertolicells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); human lung cells(W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mousemammary tumor (MMT 060562, ATCC CCL51). The selection of the appropriatehost cell is deemed to be within the skill in the art.

3. Selection and Use of a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO285, PRO286, orPRO358 may be inserted into a replicable vector for cloning(amplification of the DNA) or for expression. Various vectors arepublicly available. The vector may, for example, be in the form of aplasmid, cosmid, viral particle, or phage. The appropriate nucleic acidsequence may be inserted into the vector by a variety of procedures. Ingeneral, DNA is inserted into an appropriate restriction endonucleasesite(s) using techniques known in the art. Vector components generallyinclude, but are not limited to, one or more of a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence. Construction ofsuitable vectors containing one or more of these components employsstandard ligation techniques which are known to the skilled artisan.

The PRO285, PRO286 and PRO358 proteins may be produced recombinantly notonly directly, but also as a fusion polypeptide with a heterologouspolypeptide, which may be a signal sequence or other polypeptide havinga specific cleavage site at the N-terminus of the mature protein orpolypeptide. In general, the signal sequence may be a component of thevector, or it may be a part of the PRO285, PRO286 or PRO358 DNA that isinserted into the vector. The signal sequence may be a prokaryoticsignal sequence selected, for example, from the group of the alkalinephosphatase, penicillinase, 1 pp, or heat-stable enterotoxin II leaders.For yeast secretion the signal sequence may be, e.g., the yeastinvertase leader, alpha factor leader (including Saccharomyces andKluyveromyces α-factor leaders, the latter described in U.S. Pat. No.5,010,182), or acid phosphatase leader, the C. albicans glucoamylaseleader (EP 362,179 published 4 Apr. 1990), or the signal described in WO90/13646 published 15 Nov. 1990. In mammalian cell expression, mammaliansignal sequences may be used to direct secretion of the protein, such assignal sequences from secreted polypeptides of the same or relatedspecies, as well as viral secretory leaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2μ plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up the PRO285,PRO286, or PRO358 nucleic acid, such as DHFR or thymidine kinase. Anappropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl, Acad. Sci. USA, 77:4216 (1980). A suitableselection gene for use in yeast is the trp1 gene present in the yeastplasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1[Jones, Genetics, 85:12 (1977)].

Expression and cloning vectors usually contain a promoter operablylinked to the nucleic acid sequence encoding the PRO285, PRO286 orPRO358 protein to direct mRNA synthesis. Promoters recognized by avariety of potential host cells are well known. Promoters suitable foruse with prokaryotic hosts include the β-lactamase and lactose promotersystems [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature,281:544 (1979)], alkaline phosphatase, a tryptophan (trp) promotersystem [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], andhybrid promoters such as the tac promoter [deBoer et al., Proc. Natl.Acad. Sci. USA, 80:21-25 (1983)]. Promoters for use in bacterial systemsalso will contain a Shine-Dalgarno (S.D.) sequence operably linked tothe DNA encoding PRO285, PRO286, or PRO358.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

PRO285, PRO286 or PRO358 transcription from vectors in mammalian hostcells is controlled, for example, by promoters obtained from the genomesof viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published5 Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus,avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virusand Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g.,the actin promoter or an immunoglobulin promoter, and from heat-shockpromoters, provided such promoters are compatible with the host cellsystems.

Transcription of a DNA encoding the PRO285, PRO286, or PRO358polypeptide by higher eukaryotes may be increased by inserting anenhancer sequence into the vector. Enhancers are cis-acting elements ofDNA, usually about from 10 to 300 bp, that act on a promoter to increaseits transcription. Many enhancer sequences are now known from mammaliangenes (globin, elastase, albumin, α-fetoprotein, and insulin).Typically, however, one will use an enhancer from a eukaryotic cellvirus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. The enhancer may be spliced into thevector at a position 5′ or 3′ to the PRO285, PRO286, or PRO358 codingsequence, but is preferably located at a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding PRO285, PRO286, or PRO358.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of PRO285, PRO286, or PRO358 in recombinant vertebratecell culture are described in Gething et al., Nature, 293:620-625(1981); Mantei et al., Nature, 281:40-46 (1979); EP 117,060; and EP117,058.

4. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequencePRO285, PRO286 or PRO358 polypeptides or against a synthetic peptidebased on the DNA sequences provided herein or against exogenous sequencefused to PRO285, PRO286 or PRO358 DNA and encoding a specific antibodyepitope.

5. Purification of Polypeptide

Forms of PRO285, PRO286 or PRO358 may be recovered from culture mediumor from host cell lysates. If membrane-bound, it can be released fromthe membrane using a suitable detergent solution (e.g. Triton-X 100) orby enzymatic cleavage. Cells employed in expression of PRO285, PRO286 orPRO358 can be disrupted by various physical or chemical means, such asfreeze-thaw cycling, sonication, mechanical disruption, or cell lysingagents.

It may be desired to purify PRO285, PRO286, or PRO358 from recombinantcell proteins or polypeptides. The following procedures are exemplary ofsuitable purification procedures: by fractionation on an ion-exchangecolumn; ethanol precipitation; reverse phase HPLC; chromatography onsilica or on a cation-exchange resin such as DEAE; chromatofocusing;SDS-PAGE; ammonium sulfate precipitation; gel filtration using, forexample, Sephadex G-75; protein A Sepharose columns to removecontaminants such as IgG; and metal chelating columns to bindepitope-tagged forms of the Toll proteins. Various methods of proteinpurification may be employed and such methods are known in the art anddescribed for example in Deutscher, Methods in Enzymology, 182 (1990);Scopes, Protein Purification: Principles and Practice, Springer-Verlag,New York (1982). The purification step(s) selected will depend, forexample, on the nature of the production process used and the particularToll protein produced.

E. Uses for the Toll Proteins and Encoding Nucleic Acids

Nucleotide sequences (or their complement) encoding the Toll proteins ofthe present invention have various applications in the art of molecularbiology, including uses as hybridization probes, in chromosome and genemapping and in the generation of anti-sense RNA and DNA. Toll nucleicacid will also be useful for the preparation of PRO285, PRO286 andPRO358 polypeptides by the recombinant techniques described herein.

The full-length native sequence DNA40021, DNA42663, and DNA47361 genes,encoding PRO285, PRO286, and PRO358, respectively, or portions thereof,may be used as hybridization probes for a cDNA library to isolate thefull-length gene or to isolate still other genes (for instance, thoseencoding naturally-occurring variants of PRO285, PRO286, or PRO358 ortheir further human homologues, or homologues from other species) whichhave a desired sequence identity to the PRO285, PRO286, or PRO358sequence disclosed in FIGS. 1, 3 and 12A-B, respectively. Optionally,the length of the probes will be about 20 to about 50 bases. Thehybridization probes may be derived from the nucleotide sequence of FIG.2 (SEQ ID NO: 2), or FIG. 4 (SEQ ID NO: 4), or FIG. 13A-B (SEQ ID NO:14), or from genomic sequences including promoters, enhancer elementsand introns of native sequence. By way of example, a screening methodwill comprise isolating the coding region of the PRO285, or PRO286, orPRO358 gene using the known DNA sequence to synthesize a selected probeof about 40 bases. Hybridization probes may be labeled by a variety oflabels, including radionucleotides such as ³²P or ³⁵S, or enzymaticlabels such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems. Labeled probes having a sequencecomplementary to that of the PRO285, PRO286, or PRO358 gene (DNAs 40021,42663 and 47361) of the present invention can be used to screenlibraries of human cDNA, genomic DNA or mRNA to determine which membersof such libraries the probe hybridizes to. Hybridization techniques aredescribed in further detail in the Examples below.

The probes may also be employed in PCR techniques to generate a pool ofsequences for identification of closely related Toll sequences.

Nucleotide sequences encoding a Toll protein herein can also be used toconstruct hybridization probes for mapping the gene which encodes thatToll protein and for the genetic analysis of individuals with geneticdisorders. The nucleotide sequences provided herein may be mapped to achromosome and specific regions of a chromosome using known techniques,such as in situ hybridization, linkage analysis against knownchromosomal markers, and hybridization screening with libraries.

The human Toll proteins of the present invention can also be used inassays to identify other proteins or molecules involved in Toll-mediatedsignal transduction. For example, PRO285, PRO286, and PRO358 are usefulin identifying the as of yet unknown natural ligands of human Tolls, orother factors that participate (directly or indirectly) in theactivation of and/or signaling through a human Toll receptor, such aspotential Toll receptor associated kinases. In addition, inhibitors ofthe receptor/ligand binding interaction can be identified. Proteinsinvolved in such binding interactions can also be used to screen forpeptide or small molecule inhibitors or agonists of the bindinginteraction. Screening assays can be designed to find lead compoundsthat mimic the biological activity of a native Toll polypeptide or aligand for a native Toll polypeptide. Such screening assays will includeassays amenable to high-throughput screening of chemical libraries,making them particularly suitable for identifying small molecule drugcandidates. Small molecules contemplated include synthetic organic orinorganic compounds. The assays can be performed in a variety offormats, including protein-protein binding assays, biochemical screeningassays, immunoassays and cell based assays, which are well characterizedin the art.

In vitro assays employ a mixture of components including a Toll receptorpolypeptide, which may be part of fusion product with another peptide orpolypeptide, e.g., a tag for detecting or anchoring, etc. The assaymixtures may further comprise (for binding assays) a natural intra- orextracellular Toll binding target (i.e. a Toll ligand, or anothermolecule known to activate and/or signal through the Toll receptor).While native binding targets may be used, it is frequently preferred touse portion of such native binding targets (e.g. peptides), so long asthe portion provides binding affinity and avidity to the subject Tollprotein conveniently measurable in the assay. The assay mixture alsocontains a candidate pharmacological agent. Candidate agents encompassnumerous chemical classes, through typically they are organic compounds,preferably small organic compounds, and are obtained from a wide varietyof sources, including libraries of synthetic or natural compounds. Avariety of other reagents may also be included in the mixture, such as,salts, buffers, neutral proteins, e.g. albumin, detergents, proteaseinhibitors, nuclease inhibitors, antimicrobial agents, etc.

In in vitro binding assays, the resultant mixture is incubated underconditions whereby, but for the presence of the candidate molecule, theToll protein specifically binds the cellular binding target, portion oranalog, with a reference binding affinity. The mixture components can beadded in any order that provides for the requisite bindings andincubations may be performed at any temperature which facilitatesoptimal binding. Incubation periods are likewise selected for optimalbinding but also minimized to facilitate rapid high-throughputscreening.

After incubation, the agent-biased binding between the Toll protein andone or more binding targets is detected by any convenient technique. Forcell-free binding type assays, a separation step is often used toseparate bound from unbound components. Separation may be effected byprecipitation (e.g. TCA precipitation, immunoprecipitation, etc.),immobilization (e.g on a solid substrate), etc., followed by washing by,for example, membrane filtration (e.g. Whatman's P-18 ion exchangepaper, Polyfiltronic's hydrophobic GFC membrane, etc.), gelchromatography (e.g. gel filtration, affinity, etc.). For Toll-dependenttranscription assays, binding is detected by a change in the expressionof a Toll-dependent reporter.

Detection may be effected in any convenient way. For cell-free bindingassays, one of the components usually comprises or is coupled to alabel. The label may provide for direct detection as radioactivity,luminescence, optical or electron density, etc., or indirect detection,such as, an epitope tag, an enzyme, etc. A variety of methods may beused to detect the label depending on the nature of the label and otherassay components, e.g. through optical or electron density, radiativeemissions, nonradiative energy transfers, etc. or indirectly detectedwith antibody conjugates, etc.

Nucleic acids which encode PRO285, PRO286, or PRO358, or their modifiedforms can also be used to generate either transgenic animals or “knockout” animals which, in turn, are useful in the development and screeningof therapeutically useful reagents. A transgenic animal (e.g., a mouseor rat) is an animal having cells that contain a transgene, whichtransgene was introduced into the animal or an ancestor of the animal ata prenatal, e.g., an embryonic stage. A transgene is a DNA which isintegrated into the genome of a cell from which a transgenic animaldevelops. In one embodiment, cDNA encoding PRO285 or PRO286 can be usedto clone genomic DNA encoding PRO285, PRO286, or PRO358 in accordancewith established techniques and the genomic sequences used to generatetransgenic animals that contain cells which express DNA encoding PRO285,PRO286, or PRO358. Methods for generating transgenic animals,particularly animals such as mice or rats, have become conventional inthe art and are described, for example, in U.S. Pat. Nos. 4,736,866 and4,870,009. Typically, particular cells would be targeted for transgeneincorporation with tissue-specific enhancers. Transgenic animals thatinclude a copy of a transgene encoding PRO285, PRO286, or PRO358introduced into the germ line of the animal at an embryonic stage can beused to examine the effect of increased expression of DNA encodingPRO285, PRO286, or PRO358. Such animals can be used as tester animalsfor reagents thought to confer protection from, for example,pathological conditions associated with its overexpression. Inaccordance with this facet of the invention, an animal is treated withthe reagent and a reduced incidence of the pathological condition,compared to untreated animals bearing the transgene, would indicate apotential therapeutic intervention for the pathological condition.

Alternatively, non-human vertebrate (e.g. mammalian) homologues ofPRO285 or PRO286 or PRO358 can be used to construct a “knock out” animalwhich has a defective or altered gene encoding PRO285 or PRO286 orPRO358, as a result of homologous recombination between the endogenousgene encoding PRO285, PRO286, or PRO358 protein and altered genomic DNAencoding PRO285, PRO286, or PRO358 introduced into an embryonic cell ofthe animal. For example, cDNA encoding PRO285, PRO286, or PRO358 can beused to clone genomic DNA encoding PRO285, PRO286, or PRO358 inaccordance with established techniques. A portion of the genomic DNAencoding PRO285, PRO286, or PRO358 can be deleted or replaced withanother gene, such as a gene encoding a selectable marker which can beused to monitor integration. Typically, several kilobases of unalteredflanking DNA (both at the 5′ and 3′ ends) are included in the vector[see e.g., Thomas and Capecchi, Cell, 51:503 (1987) for a description ofhomologous recombination vectors]. The vector is introduced into anembryonic stem cell line (e.g., by electroporation) and cells in whichthe introduced DNA has homologously recombined with the endogenous DNAare selected [see e.g., Li et al., Cell, 69:915 (1992)]. The selectedcells are then injected into a blastocyst of an animal (e.g., a mouse orrat) to form aggregation chimeras [see e.g., Bradley, inTeratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J.Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. A chimeric embryo canthen be implanted into a suitable pseudopregnant female foster animaland the embryo brought to term to create a “knock out” animal. Progenyharboring the homologously recombined DNA in their germ cells can beidentified by standard techniques and used to breed animals in which allcells of the animal contain the homologously recombined DNA. Knockoutanimals can be characterized for instance, for their ability to defendagainst certain pathological conditions and for their development ofpathological conditions due to absence of the PRO285, PRO286, or PRO358polypeptides.

Nucleic acid encoding the Toll polypeptide disclosed herein may also beused in gene therapy. In gene therapy applications, genes are introducedinto cells in order to achieve in vivo synthesis of a therapeuticallyeffective genetic product, for example for replacement of a defectivegene. “Gene therapy” includes both conventional gene therapy where alasting effect is achieved by a single treatment, and the administrationof gene therapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA. AntisenseRNAs and DNAs can be used as therapeutic agents for blocking theexpression of certain genes in vivo. It has already been shown thatshort antisense oligonucleotides can be imported into cells where theyact as inhibitors, despite their low intracellular concentrations causedby their restricted uptake by the cell membrane. (Zamecnik et al., Proc.Natl. Acad. Sci. USA 83, 4143-4146 [1986]). The oligonucleotides can bemodified to enhance their uptake, e.g. by substituting their negativelycharged phosphodiester groups by uncharged groups.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. The currently preferred in vivogene transfer techniques include transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection (Dzau et al., Trends in Biotechnology 11, 205-210 [1993]).In some situations it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA 87, 3410-3414 (1990). For review of the currently knowngene marking and gene therapy protocols see Anderson et al., Science256, 808-813 (1992).

The various uses listed in connection with the Toll proteins herein, arealso available for agonists of the native Toll receptors, which mimic atleast one biological function of a native Toll receptor.

F. Anti-Toll Protein Antibodies

The present invention further provides anti-Toll protein antibodies.Exemplary antibodies include polyclonal, monoclonal, humanized,bispecific, and heteroconjugate antibodies.

1. Polyclonal Antibodies

The anti-Toll protein antibodies may comprise polyclonal antibodies.Methods of preparing polyclonal antibodies are known to the skilledartisan. Polyclonal antibodies can be raised in a mammal, for example,by one or more injections of an immunizing agent and, if desired, anadjuvant. Typically, the immunizing agent and/or adjuvant will beinjected in the mammal by multiple subcutaneous or intraperitonealinjections. The immunizing agent may include the PRO285 and PRO286polypeptides or a fusion protein thereof. It may be useful to conjugatethe immunizing agent to a protein known to be immunogenic in the mammalbeing immunized. Examples of such immunogenic proteins include but arenot limited to keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, and soybean trypsin inhibitor. Examples of adjuvantswhich may be employed include Freund's complete adjuvant and MPL-TDMadjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).The immunization protocol may be selected by one skilled in the artwithout undue experimentation.

2. Monoclonal Antibodies

The anti-Toll protein antibodies may, alternatively, be monoclonalantibodies. Monoclonal antibodies may be prepared using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975). In a hybridoma method, a mouse, hamster, or other appropriatehost animal, is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The immunizing agent will typically include the PRO285, PRO286, orPRO358 polypeptides or a fusion protein thereof. Generally, eitherperipheral blood lymphocytes (“PBLs”) are used if cells of human originare desired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell [Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986) pp. 59-103].Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells may becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (“HAT medium”), which substances prevent thegrowth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Rockville, Md. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed againstPRO285, PRO286, or PRO358. Preferably, the binding specificity ofmonoclonal antibodies produced by the hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods[Goding, supra]. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences [U.S. Pat.No. 4,816,567; Morrison et al., supra] or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart.

3. Humanized and Human Antibodies

The anti-Toll antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991). Similarly,human antibodies can be made by introducing of human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995).

4. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities may befor the PRO285, PRO286, or PRO358 protein, the other one for any otherantigen, and preferably for a cell-surface protein or receptor orreceptor subunit. It is also possible to prepare bispecific antibodies,having specificities to two different Toll-like proteins, such as, anytwo of the Toll homologues disclosed in the present application, or aToll protein disclosed herein, and a Toll protein known in the art,e.g., TLR2. Such bispecific antibodies could block the recognition ofdifferent pathogen patterns by Toll receptors, and are, therefore,expected to have significant benefits in the treatment of sepsis andseptic shock.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities [Milsteinand Cuello, Nature, 305:537-539 (1983)]. Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

5. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells [U.S. Pat. No. 4,676,980],and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP03089]. It is contemplated that the antibodies may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

G. Uses for Anti-Toll Protein Antibodies

The anti-Toll antibodies of the invention have various utilities. Forexample, anti-PRO285, anti-PRO286, anti-PRO-358, and anti-TLR2antibodies may be used in diagnostic assays for PRO285, PRO286, PRO358,or TLR2 e.g., detecting its expression in specific cells, tissues, orserum. Various diagnostic assay techniques known in the art may be used,such as competitive binding assays, direct or indirect sandwich assaysand immunoprecipitation assays conducted in either heterogeneous orhomogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques,CRC Press, Inc. (1987) pp. 147-158]. The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase. Any method known in theart for conjugating the antibody to the detectable moiety may beemployed, including those methods described by Hunter et al., Nature,144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al.,J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. andCytochem., 30:407 (1982).

Anti-PRO285, anti-PRO286, anti-PRO358, or anti-TLR2 antibodies also areuseful for the affinity purification of these proteins from recombinantcell culture or natural sources. In this process, the antibodies againstthese Troll proteins are immobilized on a suitable support, such aSephadex resin or filter paper, using methods well known in the art. Theimmobilized antibody then is contacted with a sample containing the tobe purified, and thereafter the support is washed with a suitablesolvent that will remove substantially all the material in the sampleexcept the PRO285, PRO286, PRO358, or TLR2 protein which is bound to theimmobilized antibody. Finally, the support is washed with anothersuitable solvent that will release the protein from the antibody.

Anti-Toll receptor (i.e., anti-PRO285, anti-PRO286, anti-PRO358, oranti-TLR2 antibodies) may also be useful in blocking the biologicalactivities of the respective Toll receptors. The primary function of thefamily of Toll receptors is believed to be to act as pathogen patternrecognition receptors sensing the presence of conserved molecularpattern present on microbes. Lipopolysaccharides (LPS, also known asendotoxins), potentially lethal molecules produced by various bacteria,bind to the lipopolysaccharide binding protein (LBP) in the blood. Thecomplex formed then activates a receptor known as CD14. There is noconsensus in the art about what happens next. According to a hypothesis,CD14 does not directly instruct macrophages to produce cytokines, celladhesion proteins and enzymes involved in the production of lowermolecular weight proinflammatory mediators, rather enables LPS toactivate a second receptor. Alternatively, it has been suggested thatLPS may activate certain receptors directly, without help from LBP orCD14. The data disclosed in the present application indicate that thehuman toll-like receptors are signaling receptors that are activated byLPS in an LBP and CD14 responsive manner. As this mechanism, underpathophysiologic conditions can lead to an often fatal syndrome calledseptic shock, anti-Toll receptor antibodies (just as other Toll receptorantagonists) might be useful in the treatment of septic shock. It isforeseen that the different Toll receptors might recognize differentpathogens, e.g., various strains of Gram-negative or Gram-positivebacteria. Accordingly, in certain situations, combination therapy with amixture of antibodies specifically binding different Toll receptors, orthe use of bispecific anti-Toll antibodies may be desirable.

It is specifically demonstrated herein that anti-huTLR2 antibodies arebelieved to be specifically useful in blocking the induction of thisreceptor by LPS. As it has been shown that LPS exposure can lead toseptic shock (Parrillo, N. Engl. J. Med. 328, 1471-1477 [1993]),anti-huTLR2 antibodies are potentially useful in the treatment of septicshock.

The foregoing therapeutic and diagnostic uses listed in connection withthe anti-Toll receptor antibodies are also applicable to other Tollantagonists, i.e., other molecules (proteins, peptides, small organicmolecules, etc.) that block Toll receptor activation and/or signaltransduction mediated by Toll receptors.

In view of their therapeutic potentials, the Toll proteins (includingvariants of the native Toll homologues), and their agonists andantagonists (including but not limited to anti-Toll antibodies) areincorporated in compositions suitable for therapeutic use. Therapeuticcompositions are prepared for storage by mixing the active ingredienthaving the desired degree of purity with optional physiologicallyacceptable carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences 16th Edition, Osol, A. Ed. 1980) in the form oflyophilized formulations or aqueous solutions. Acceptable carriers,excipients or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrateand other organic acids; antioxidants including ascorbic acid; lowmolecular weight (less than about 10 residues) polypeptides; proteins,such as serum albumin, gelatin or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidone, amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides and othercarbohydrates including glucose, mannose, or dextrins; chelating agentssuch as EDTA; sugar alcohols such as mannitol or sorbitol; salt-formingcounterions such as sodium; and/or nonionic surfactants such as Tween,Pluronics or PEG.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to or following lyophilization and reconstitution.

Therapeutic compositions herein generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is in accord with known methods, e.g.injection or infusion by intravenous, intraperitoneal, intracerebral,intramuscular, intraocular, intraarterial or intralesional routes,topical administration, or by sustained release systems.

Suitable examples of sustained release preparations includesemipermeable polymer matrices in the form of shaped articles, e.g.films, or microcapsules. Sustained release matrices include polyesters,hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymersof L-glutamic acid and gamma ethyl-L-glutamate (U. Sidman et. al.,Biopolymers 22 (1): 547-556 [1983]), poly (2-hydroxyethyl-methacrylate)(R. Langer, et al., J. Biomed. Mater. Res. 15: 167-277 [1981] and R.Langer, Chem. Tech. 12: 98-105 [1982]), ethylene vinyl acetate (R.Langer et al., Id.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988).Sustained release compositions also include liposomes. Liposomescontaining a molecule within the scope of the present invention areprepared by methods known per se: DE 3,218,121; Epstein et al., Proc.Natl. Acad. Sci. USA 82: 3688-3692 (1985); Hwang et al., Proc. Natl.Acad. Sci, USA 77: 4030-4034 (1980); EP 52322; EP 36676A; EP 88046; EP143949; EP 142641; Japanese patent application 83-118008; U.S. Pat. Nos.4,485,045 and 4,544,545; and EP 102,324. Ordinarily the liposomes are ofthe small (about 200-800 Angstroms) unilamelar type in which the lipidcontent is greater than about 30 mol. % cholesterol, the selectedproportion being adjusted for the optimal NT-4 therapy.

An effective amount of the active ingredient will depend, for example,upon the therapeutic objectives, the route of administration, and thecondition of the patient. Accordingly, it will be necessary for thetherapist to titer the dosage and modify the route of administration asrequired to obtain the optimal therapeutic effect. A typical dailydosage might range from about 1 μg/kg to up to 100 mg/kg or more,depending on the factors mentioned above. Typically, the clinician willadminister a molecule of the present invention until a dosage is reachedthat provides the required biological effect. The progress of thistherapy is easily monitored by conventional assays.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Rockville, Md.

Example 1 Isolation of cDNA Clones Encoding Human PRO285

A proprietary expressed sequence tag (EST) DNA database (LIFESEQ™,Incyte Pharmaceuticals, Palo Alto, Calif.) was searched and an EST(#2243209) was identified which showed homology to the Drosophila Tollprotein.

Based on the EST, a pair of PCR primers (forward and reverse):

TAAAGACCCAGCTGTGACCG (SEQ ID NO: 5) ATCCATGAGCCTCTGATGGG, (SEQ ID NO: 6)anda probe:

(SEQ ID NO: 7) ATTTATGTCTCGAGGAAAGGGACTGGTTACCAGGGCAGCCAGTTCwere synthesized.

mRNA for construction of the cDNA libraries was isolated from humanplacenta tissue. The cDNA libraries used to isolate the cDNA clones wereconstructed by standard methods using commercially available reagentssuch as those from Invitrogen, San Diego, Calif. (Fast Track 2). ThecDNA was primed with oligo dT containing a NotI site, linked with bluntto SalI hemikinased adaptors, cleaved with NotI, sized appropriately bygel electrophoresis, and cloned in a defined orientation into thecloning vector pCR2.1 (Invitrogen, Inc.) using reagents and protocolsfrom Life Technologies, Gaithersburg, Md. (Super Script Plasmid System).The double stranded cDNA was sized to greater than 1000 bp and the cDNAwas cloned into BamHI/NotI cleaved vector. pCR2.1 is a commerciallyavailable plasmid, designed for easy cloning of PCR fragments, thatcarries AmpR and KanR genes for selection, and LacZ gene for blue-whiteselection.

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO285 gene using the probe oligonucleotideand one of the PCR primers.

A cDNA clone was sequenced in entirety. The entire nucleotide sequenceof DNA40021 (encoding PRO285) is shown in FIG. 2 (SEQ ID NO:2). CloneDNA40021 contains a single open reading frame with an apparenttranslational initiation site at nucleotide positions 61-63 (FIG. 2).The predicted polypeptide precursor is 1049 amino acids long, includinga putative signal peptide at amino acid positions 1-29, a putativetransmembrane domain between amino acid positions 837-860, and a leucinezipper pattern at amino acid positions 132-153 and 704-725,respectively. It is noted that the indicated boundaries are approximate,and the actual limits of the indicated regions might differ by a fewamino acids. Clone DNA40021 has been deposited with ATCC (designation:DNA40021-1154) and is assigned ATCC deposit no. 209389.

Based on a BLAST and FastA sequence alignment analysis (using the ALIGNcomputer program) of the full-length sequence is a human analogue of theDrosophila Toll protein, and is homologous to the following human Tollproteins: Toll1 (DNAX# HSU88540-1, which is identical with the randomsequenced full-length cDNA #HUMRSC786-1); Toll2 (DNAX# HSU88878-1);Toll3 (DNAX# HSU88879-1); and Toll4 (DNAX# HSU88880-1).

Example 2 Isolation of cDNA Clones Encoding Human PRO286

A proprietary expressed sequence tag (EST) DNA database (LIFESEQ™,Incyte Pharmaceuticals, Palo Alto, Calif.) was searched and an EST(#694401) was identified which showed homology to the Drosophila Tollprotein.

Based on the EST, a pair of PCR primers (forward and reverse):

GCCGAGACAAAAACGTTCTCC (SEQ ID NO: 8) CATCCATGTTCTCATCCATTAGCC, (SEQ IDNO: 9)anda probe:

(SEQ ID NO: 10) TCGACAACCTCATGCAGAGCATCAACCAAAGCAAGAAAACAGTATTwere synthesized.

mRNA for construction of the cDNA libraries was isolated from humanplacenta tissue. This RNA was used to generate an oligo dT primed cDNAlibrary in the vector pRK5D using reagents and protocols from LifeTechnologies, Gaithersburg, Md. (Super Script Plasmid System). pRK5D isa cloning vector that has an sp6 transcription initiation site followedby an SfiI restriction enzyme site preceding the XhoI/NotI cDNA cloningsites. The cDNA was primed with oligo dT containing a NotI site, linkedwith blunt to SalI hemikinased adaptors, cleaved with NotI, sized togreater than 1000 by appropriately by gel electrophoresis, and cloned ina defined orientation into XhoI/NotI-cleaved pRK5D.

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO286 gene using the probe oligonucleotideidentified above and one of the PCR primers.

A cDNA clone was sequenced in entirety. The entire nucleotide sequenceof DNA42663 (encoding PRO286) is shown in FIG. 4 (SEQ ID NO:4). CloneDNA42663 contains a single open reading frame with an apparenttranslational initiation site at nucleotide positions 57-59 (FIG. 4).The predicted polypeptide precursor is 1041 amino acids long, includinga putative signal peptide at amino acid positions 1-26, a potentialtransmembrane domain at amino acid positions 826-848, and leucine zipperpatterns at amino acids 130-151, 206-227, 662-684, 669-690 and 693-614,respectively. It is noted that the indicated boundaries are approximate,and the actual limits of the indicated regions might differ by a fewamino acids.

Clone DNA42663 has been deposited with ATCC (designation: DNA42663-1154)and is assigned ATCC deposit no. 209386.

Based on a BLAST and FastA sequence alignment analysis (using the ALIGNcomputer program) of the full-length sequence of PRO286, it is a humananalogue of the Drosophila Toll protein, and is homologous to thefollowing human Toll proteins: Toll1 (DNAX# HSU88540-1, which isidentical with the random sequenced full-length cDNA #HUMRSC786-1);Toll2 (DNAX# HSU88878-1); Toll3 (DNAX# HSU88879-1); and Toll4 (DNAX#HSU88880-1).

Example 3 Isolation of cDNA Clones Encoding Human PRO358

The extracellular domain (ECD) sequences (including the secretion signalsequence, if any) from known members of the human Toll receptor familywere used to search EST databases. The EST databases included public ESTdatabases (e.g., GenBank) and a proprietary EST database (LIFESEQ™,Incyte Pharmaceuticals, Palo Alto, Calif.). The search was performedusing the computer program BLAST or BLAST2 [Altschul et al., Methods inEnzymology, 266:460-480 (1996)] as a comparison of the ECD proteinsequences to a 6 frame translation of the EST sequences. Thosecomparisons resulting in a BLAST score of 70 (or in some cases, 90) orgreater that did not encode known proteins were clustered and assembledinto consensus DNA sequences with the program “phrap” (Phil Green,University of Washington, Seattle, Wash.).

An EST was identified in the Incyte database (INC3115949).

Based on the EST sequence, oligonucleotides were synthesized to identifyby PCR a cDNA library that contained the sequence of interest and foruse as probes to isolate a clone of the full-length coding sequence forPRO358.

A pair of PCR primers (forward and reverse) were synthesized:

TCCCACCAGGTATCATAAACTGAA (SEQ ID NO: 15) TTATAGACAATCTGTTCTCATCAGAGA(SEQ ID NO: 16)A probe was also synthesized:

(SEQ ID NO: 17) AAAAAGCATACTTGGAATGGCCCAAGGATAGGTGTAAATGIn order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO358 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human bonemarrow (LIB256). The cDNA libraries used to isolated the cDNA cloneswere constructed by standard methods using commercially availablereagents such as those from Invitrogen, San Diego, Calif. The cDNA wasprimed with oligo dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO358 (FIGS. 13A and 13B, SEQ ID NO:14)and the derived protein sequence for PRO358 (FIGS. 12A-12C, SEQ IDNO:13).

The entire nucleotide sequence of the clone identified (DNA47361) isshown in FIG. 13A-B (SEQ ID NO:14). Clone DNA47361 contains a singleopen reading frame with an apparent translational initiation site (ATGstart signal) at nucleotide positions underlined in FIGS. 13A and 13B.The predicted polypeptide precursor is 811 amino acids long, including aputative signal sequence (amino acids 1 to 19), an extracellular domain(amino acids 20 to 575, including leucine rich repeats in the regionfrom position 55 to position 575), a putative transmembrane domain(amino acids 576 to 595). Clone DNA47361 (designated DNA47361-1249) hasbeen deposited with ATCC and is assigned ATCC deposit, no. 209431.

Based on a BLAST and FastA sequence alignment analysis (using the ALIGNcomputer program) of the full-length sequence of PRO286, it is a humananalogue of the Drosophila Toll protein, and is homologous to thefollowing human Toll proteins: Toll1. (DNAX# HSU88540-1, which isidentical with the random sequenced full-length cDNA #HUMRSC786-1);Toll2 (DNAX# HSU88878-1); Toll3 (DNAX# HSU88879-1); and Toll4 (DNAX#HSU88880-1).

Example 4 Use of PRO285, PRO286 and PRO358 DNA as a Hybridization Probe

The following method describes use of a nucleotide sequence encodingPRO285, PRO286 or PRO358 as a hybridization probe. In the followingdescription, these proteins are collectively referred to as “Tollhomologues.”

DNA comprising the coding sequence of a Toll homologue is employed as aprobe to screen for homologous DNAs (such as those encodingnaturally-occurring variants of these particular Toll proteins in humantissue cDNA libraries or human tissue genomic libraries.

Hybridization and washing of filters containing either library DNAs isperformed under the following high stringency conditions. Hybridizationof radiolabeled Toll homologue-derived probe to the filters is performedin a solution of 50% formamide, 5×SSC, 0.1% SDS, 0.1% sodiumpyrophosphate, 50 mM sodium phosphate, pH 6.8, 2×Denhardt's solution,and 10% dextran sulfate at 42° C. for 20 hours. Washing of the filtersis performed in an aqueous solution of 0.1×SSC and 0.1% SDS at 42° C.

DNAs having a desired sequence identity with the DNA encodingfull-length native sequence Toll homologue can then be identified usingstandard techniques known in the art.

Example 5 Expression of PRO285, PRO286, and PRO358 in E. coli

This example illustrates preparation of an unglycosylated form ofPRO285, PRO285 or PRO358 (“Toll homologues”) by recombinant expressionin E. coli.

The DNA sequence encoding a Toll homologue is initially amplified usingselected PCR primers. The primers should contain restriction enzymesites which correspond to the restriction enzyme sites on the selectedexpression vector. A variety of expression vectors may be employed. Anexample of a suitable vector is pBR322 (derived from E. coli; seeBolivar et al., Gene, 2:95 (1977)) which contains genes for ampicillinand tetracycline resistance. The vector is digested with restrictionenzyme and dephosphorylated. The PCR amplified sequences are thenligated into the vector. The vector will preferably include sequenceswhich encode for an antibiotic resistance gene, a trp promoter, apolyhis leader (including the first six STII codons, polyhis sequence,and enterokinase cleavage site), the PRO285 coding region, lambdatranscriptional terminator, and an argU gene.

The ligation mixture is then used to transform a selected E. coli strainusing the methods described in Sambrook et al., supra. Transformants areidentified by their ability to grow on LB plates and antibioticresistant colonies are then selected. Plasmid DNA can be isolated andconfirmed by restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid culture medium such asLB broth supplemented with antibiotics. The overnight culture maysubsequently be used to inoculate a larger scale culture. The cells arethen grown to a desired optical density, during which the expressionpromoter is turned on.

After culturing the cells for several more hours, the cells can beharvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized Toll homologue can then be purified using a metalchelating column under conditions that allow tight binding of theprotein.

Example 6 Expression of PRO285, PRO286 and PRO358 in Mammalian Cells

This example illustrates preparation of a glycosylated form of PRO285,PRO286 and PRO358 (“Toll homologues”) by recombinant expression inmammalian cells.

The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employedas the expression vector. Optionally, the Toll homologue-encoding DNA isligated into pRK5 with selected restriction enzymes to allow insertionof the Toll homologue-encoding DNA using ligation methods such asdescribed in Sambrook et al., supra. The resulting vector is calledpRK5-PRO285, -PRO286 or -PRO358, as the case may be.

In one embodiment, the selected host cells may be 293 cells. Human 293cells (ATCC CCL 1573) are grown to confluence in tissue culture platesin medium such as DMEM supplemented with fetal calf serum andoptionally, nutrient components and/or antibiotics. About 10 μgpRK5-PRO285, -PRO286, or -PRO358 DNA is mixed with about 1 μg DNAencoding the VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)] anddissolved in 500 μl of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl₂. Tothis mixture is added, dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mMNaCl, 1.5 mM NaPO₄, and a precipitate is allowed to form for 10 minutesat 25° C. The precipitate is suspended and added to the 293 cells andallowed to settle for about four hours at 37° C. The culture medium isaspirated off and 2 ml of 20% glycerol in PBS is added for 30 seconds.The 293 cells are then washed with serum free medium, fresh medium isadded and the cells are incubated for about 5 days.

Approximately 24 hours after the transfections, the culture medium isremoved and replaced with culture medium (alone) or culture mediumcontaining 200 μCi/ml ³⁵S-cysteine and 200 μCi/ml ³⁵S-methionine. Aftera 12 hour incubation, the conditioned medium is collected, concentratedon a spin filter, and loaded onto a 15% SDS gel. The processed gel maybe dried and exposed to film for a selected period of time to reveal thepresence of PRO285 polypeptide. The cultures containing transfectedcells may undergo further incubation (in serum free medium) and themedium is tested in selected bioassays.

In an alternative technique, Toll homologue DNA may be introduced into293 cells transiently using the dextran sulfate method described bySomparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells aregrown to maximal density in a spinner flask and 700 μgpRK5-PRO(285)/(286)/(358) DNA is added. The cells are first concentratedfrom the spinner flask by centrifugation and washed with PBS. TheDNA-dextran precipitate is incubated on the cell pellet for four hours.The cells are treated with 20% glycerol for 90 seconds, washed withtissue culture medium, and re-introduced into the spinner flaskcontaining tissue culture medium, 5 μg/ml bovine insulin and 0.1 μg/mlbovine transferrin. After about four days, the conditioned media iscentrifuged and filtered to remove cells and debris. The samplecontaining the corresponding expressed Toll homologue can then beconcentrated and purified by any selected method, such as dialysisand/or column chromatography.

In another embodiment, the Toll homologues can be expressed in CHOcells. The pRK5-vectors can be transfected into CHO cells using knownreagents such as CaPO₄ or DEAE-dextran. As described above, the cellcultures can be incubated, and the medium replaced with culture medium(alone) or medium containing a radiolabel such as ³⁵S-methionine. Afterdetermining the presence of PRO285, PRO286 or PRO358 polypeptide, theculture medium may be replaced with serum free medium. Preferably, thecultures are incubated for about 6 days, and then the conditioned mediumis harvested. The medium containing the expressed Toll homologue canthen be concentrated and purified by any selected method.

Epitope-tagged Toll homologues may also be expressed in host CHO cells.The Toll homologue DNA may be subcloned out of the pRK5 vector. Thesubclone insert can undergo PCR to fuse in frame with a selected epitopetag such as a poly-his tag into a Baculovirus expression vector. Thepoly-his tagged insert can then be subcloned into a SV40 driven vectorcontaining a selection marker such as DHFR for selection of stableclones. Finally, the CHO cells can be transfected (as described above)with the SV40 driven vector. Labeling may be performed, as describedabove, to verify expression. The culture medium containing the expressedpoly-His tagged Toll homologue can then be concentrated and purified byany selected method, such as by Ni²⁺-chelate affinity chromatography.

Example 7 Expression of PRO285. PRO286, and PRO358 in Yeast

The following method describes recombinant expression of PRO285, PRO286or PRO358 (“Toll homologues”) in yeast.

First, yeast expression vectors are constructed for intracellularproduction or secretion of a Toll homologue from the ADH2/GAPDHpromoter. DNA encoding the desired Toll homologue, a selected signalpeptide and the promoter is inserted into suitable restriction enzymesites in the selected plasmid to direct intracellular expression. Forsecretion, DNA encoding the selected Toll homologue can be cloned intothe selected plasmid, together with DNA encoding the ADH2/GAPDHpromoter, the yeast alpha-factor secretory signal/leader sequence, andlinker sequences (if needed) for expression.

Yeast cells, such as yeast strain AB110, can then be transformed withthe expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

Recombinant Toll homologues can subsequently be isolated and purified byremoving the yeast cells from the fermentation medium by centrifugationand then concentrating the medium using selected cartridge filters. Theconcentrate containing the Toll homologue may further be purified usingselected column chromatography resins.

Example 8 Expression of PRO285, PRO286 and PRO358 in BaculovirusInfected Insects Cells

The following method describes recombinant expression of PRO285, PRO286and PRO358 (“Toll homologues”) in Baculovirus infected insect cells.

The Toll homologue coding sequence is fused upstream of an epitope tagcontained with a baculovirus expression vector. Such epitope tagsinclude poly-his tags and immunoglobulin tags (like Fc regions of IgG).A variety of plasmids may be employed, including plasmids derived fromcommercially available plasmids such as pVL1393 (Novagen). Briefly, theToll homologue coding sequence or the desired portion of the codingsequence (such as the sequence encoding the extracellular domain) isamplified by PCR with primers complementary to the 5′ and 3′ regions.The 5′ primer may incorporate flanking (selected) restriction enzymesites. The product is then digested with those selected restrictionenzymes and subcloned into the expression vector.

Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4-5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression is performed as described byO'Reilley et al., Baculovirus expression vectors: A laboratory Manual,Oxford: Oxford University Press (1994).

Expressed poly-his tagged Toll homologue can then be purified, forexample, by Ni²⁺-chelate affinity chromatography as follows. Extractsare prepared from recombinant virus-infected Sf9 cells as described byRupert et al., Nature, 362:175-179 (1993). Briefly, Sf9 cells arewashed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mMMgCl₂; 0.1 mM EDTA; 10% Glycerol; 0.1% NP-40; 0.4 M KCl), and sonicatedtwice for 20 seconds on ice. The sonicates are cleared bycentrifugation, and the supernatant is diluted 50-fold in loading buffer(50 mM phosphate, 300 mM NaCl, 10% Glycerol, pH 7.8) and filteredthrough a 0.45 μm filter. A Ni²⁺-NTA agarose column (commerciallyavailable from Qiagen) is prepared with a bed volume of 5 mL, washedwith 25 mL of water and equilibrated with 25 mL of loading buffer. Thefiltered cell extract is loaded onto the column at 0.5 mL per minute.The column is washed to baseline A₂₈₀ with loading buffer, at whichpoint fraction collection is started. Next, the column is washed with asecondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% Glycerol, pH6.0), which elutes nonspecifically bound protein. After reaching A₂₈₀baseline again, the column is developed with a 0 to 500 mM Imidazolegradient in the secondary wash buffer. One mL fractions are collectedand analyzed by SDS-PAGE and silver staining or western blot withNi²⁺-NTA-conjugated to alkaline phosphatase (Qiagen). Fractionscontaining the eluted His₁₀-tagged PRO285 are pooled and dialyzedagainst loading buffer.

Alternatively, purification of the IgG tagged (or Fc tagged) Tollhomologues can be performed using known chromatography techniques,including for instance, Protein A or protein G column chromatography.

Example 9 NF-κB Assay

As the Toll proteins signal through the NF-κB pathway, their biologicalactivity can be tested in an NF-κB assay. In this assay Jurkat cells aretransiently transfected using Lipofectamine reagent (Gibco BRL)according to the manufacturer's instructions. 1 μg pB2XLuc plasmid,containing NF-κB-driven luciferase gene, is contransfected with 1 μgpSRαN expression vector with or without the insert encoding PRO285 orPRO286. For a positive control, cells are treated with PMA (phorbolmyristyl acetate; 20 ng/ml) and PHA (phytohaemaglutinin, 2 μg/ml) forthree to four hours. Cells are lysed 2 or 3 days later for measurementof luciferase activity using reagents from Promega.

Example 10 Preparation of Antibodies that Bind PRO285. PRO286, or PRO358

This example illustrates preparation of monoclonal antibodies which canspecifically bind PRO285, PRO286 or PRO358 (“Toll homologues”).

Techniques for producing the monoclonal antibodies are known in the artand are described, for instance, in Goding, supra. Immunogens that maybe employed include purified Toll homologues, fusion proteins containingthe desired Toll homologue, and cells expressing recombinant Tollhomologues on the cell surface. Selection of the immunogen can be madeby the skilled artisan without undue experimentation.

Mice, such as Balb/c, are immunized with the Toll homologue immunogenemulsified in complete Freund's adjuvant and injected subcutaneously orintraperitoneally in an amount from 1-100 micrograms. Alternatively, theimmunogen is emulsified in MPL-TDM adjuvant (Ribi ImmunochemicalResearch, Hamilton, Mont.) and injected into the animal's hind footpads. The immunized mice are then boosted 10 to 12 days later withadditional immunogen emulsified in the selected adjuvant. Thereafter,for several weeks, the mice may also be boosted with additionalimmunization injections. Serum samples may be periodically obtained fromthe mice by retro-orbital bleeding for testing in ELISA assays to detectPRO285 antibodies.

After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of a Toll homologue. Three to four days later, the mice aresacrificed and the spleen cells are harvested. The spleen cells are thenfused (using 35% polyethylene glycol) to a selected murine myeloma cellline such as P3X63AgU.1, available from ATCC, No. CRL 1597. The fusionsgenerate hybridoma cells which can then be plated in 96 well tissueculture plates containing HAT (hypoxanthine, aminopterin, and thymidine)medium to inhibit proliferation of non-fused cells, myeloma hybrids, andspleen cell hybrids.

The hybridoma cells will be screened in an ELISA for reactivity againstthe corresponding Toll homologue. Determination of “positive” hybridomacells secreting the desired monoclonal antibodies against a Tollhomologue is within the skill in the art.

The positive hybridoma cells can be injected intraperitoneally intosyngeneic Balb/c mice to produce ascites containing the anti-Tollhomologue monoclonal antibodies. Alternatively, the hybridoma cells canbe grown in tissue culture flasks or roller bottles. Purification of themonoclonal antibodies produced in the ascites can be accomplished usingammonium sulfate precipitation, followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can be employed.

Example 11 HuTLR2 Mediates Linopolysaccharide (LPS) Induced CellularSignaling

Methods

Reagents [³H]-labeled, unlabeled, LCD25 and S. minnesota R595LPS werefrom List Biochemicals (Campbell, Calif.) and all other LPS were fromSigma Chemical Co. (St. Louis, Mo.). LP was supplied as conditionedmedia from 293 cells transfected with a human LBP expression vector. TheTLR2-Fc fusion protein was produced by baculovirus system, and purifiedas described. Mark et al., J. Biol. Chem. 269, 10720-10728 (1994).

Construction of Expression Plasmids A cDNA encoding human TLR2 wascloned from human fetal lung library. The predicted amino acid sequencematched that of the previously published sequence (Rock et al., supra),with the exception of a glu to asp substitution at amino acid 726. Theamino acid terminal epitope tag version of TLR2 (dG.TLR2) wasconstructed by adding an XhoI restriction site immediately upstream ofleucine at position 17 (the first amino acid of the predicted matureform of TLR2) and linking this to amino acids 1-53 of herpes simplexvirus type I glycoprotein D as described. Mark et al., supra. PCRproducts were sequenced and subcloned into a mammalian expression vectorthat contains the puromycin resistance gene. C-terminal truncationvariants of gD.TLR2 were constructed by digestion of the cDNA at eithera BlpI (variant Δ1) or NsiI (variant Δ2) site present in the codingsequence of the intracellular domain and at a NotI site present in the3′ polylinker of the expression vector followed by ligation ofoligonucleotide linkers.

Δ1: 5′-TCA GCG GTA AGC-3′ (SEQ ID NO: 18) and 5′-GGC CGC TTA CCG C-3′(SEQ ID NO: 19) Δ2: 5′-TAA GCT TAA CG-3′ (SEQ ID NO: 20) and 5′-GGC CGCTTA AGC TTA TGC (SEQ ID NO: 21) A-3′.

The CD4/TLR2 chimera was constructed by PCR and contained amino acids1-205 (the signal peptide and two immunoglobulin-like domains) of humanCD4 fused to amino acids 588-784 (the transmembrane and intracellulardomain) of human TLR2 with a linker-encoded valine at the junction ofthe CD4 and TLR2 sequences. The pGL3.ELAM.tk reporter plasmid containedthe sequence

(SEQ ID NO: 22) 5′-GGT ACC TTC TGA CAT CAT TGT AAT TTT AAG CAT CGT GGATAT TCC CGG GAA AGT TTT TGG ATG CCA TTG GGG ATT TCC TCT TTA GAT CTG GCGCGG TCC CAG GTC CAC TTC GCA TAT TAA GGT GAC GCG TGT GGC CTC GAA CAC CGAGCG ACC CTG CAG CGA CCC GCA AGC TT-3′,inserted between the Sad and HindIII sites of the luciferase reportedplasmid pGL3 (Promega). The C-terminal epitope tag version of LBP(LBP-FLAG) was constructed by PCR through the addition of an Asc1 sitein place of the native stop codon and the subcloning of this fragmentinto pRK5-FLAG resulting in the C-terminal addition of amino acids GRADYK DDD DK (SEQ ID NO: 23).

Stable cell lines/pools 293 human embryonic kidney cells were grown inLGDMEM/HAM's F12 (50:50) media supplemented with 10% FBS, 2 mMglutamine, and penicillin/streptomycin. For stable expression ofgD.TLR2, cells were transfected with the gD.TLR2 expression vector andselected for puromycin resistance at a final concentration of 1 μg/ml. Astable pool of cells (293-TLR2 pop1) was isolated by FACS using anantibody to the gD tag. Both the pool and the single cell clone(293-TLR2 clone 1) were characterized by FACS and western blot analysesas described in Mark et al., supra.

Luciferase reporter assay and electrophoretic mobility shift assay(EMSA) 29332 parental or stable cells (2×105 cells per well) were seededinto six-well plates, and transfected on the following day with theexpression plasmids together with 0.5 μg of the luciferase reporterplasmid pGL3-ELAM.tk and 0.05 μg of the Renilla luciferase reportedvector as an internal control. After 24 hours, cells were treated witheither LPS, LBP or both LPS and LBP and reporter gene activity wasmeasured. Data are expressed as relative luciferase activity by dividingfirefly luciferase activity with that of Renilla luciferase. For EMSA,nuclear extracts were prepared and used in a DNA-binding reaction with a5′-[³²P]-radiolabelled oligonucleotides containing a consensus NF-κBbinding site (Santa Cruz Biotechnology, sc-2511). The identity of NF-κBin the complex was confirmed by supershift with antibodies to NF-κB(data not shown).

RNA expression The tissue northern blot was purchased from Clontech andhybridized with a probe encompassing the extracellular domain of TLR2.Polyadenylated mRNA was isolated from 293 cells or 293-TLR2 cells andNorther blots were probed with human IL-8 cDNA fragment. TLR2 expressionwas determined using quantitative PCR using real time “Taqman™”technology and analyzed on a Model 770 Sequence Detector (AppliedBiosystems, Foster City, Calif., USA) essentially as described (Luoh etal., J. Mol. Endocrinol. 18, 77-85 [1997]). Forward and reverse primers,

SEQ ID NO: 24 5′-GCG GGA AGG ATT TTG GGT AA-3′, and SEQ ID NO: 25 5′-GATCCC AAC TAG ACA AAG ACT GGT C-3′were used with a hybridization probe,

SEQ ID NO: 25 5′-TGA GAG CTG CGA TAA AGT CCT AGG TTC CCA TAT-3′labeled on the 5′ nucleotide with a reporter dye FAM and the 3′nucleotide with a quenching dye TAMRA. Macrophage/monocytes were treated16 h with 1 μg/ml of LPS.

Receptor binding assay To determine the direct binding, 20 ng of[³H]-LPS was mixed with 600 ng of TLR2-Fc in 100 μl of binding buffer(150 mM NaCl, 20 mM Hepes, 0.03% BSA) containing 15 μl protein Asepharose. After 3 h-incubation at room temperature, protein A sepharosesamples were washed twice with cold PBS/0.1% NP-40 and resuspended inbinding buffer including 1% SDS and 25 mM EDTA, and counted.

Results

In Drosophila, the Toll receptor is required for embryonic dorso-ventralpattern formation and also participated in an anti-fungal immuneresponse in the adult fly. Belvin and Anderson, Ann. Rev. Cell. Biol.12, 393-416 (1996); Lemaitre et al., Cell 86, 973-983 (1996). Toll is atype I transmembrane protein containing an extracellular domain withmultiple leucine-rich repeats (LRRs) and a cytoplasmic domain withsequence homology to the interleukin-1 receptor (IL-1R), and severalplant disease-resistance proteins. Activation of Toll leads to inductionof genes through the activation of the NF-κB pathway. As noted before,there are several human homologues that have been cloned, some of whichare disclosed as novel proteins in the present application. These humanproteins mirror the topographic structure of their Drosophilacounterpart. Overexpression of a constitutively active mutant of onehuman TLR (TLR4) has been shown to lead to the activation of NF-κB andinduction of the inflammatory cytokines and constimulatory molecules(Medzhitov et al., and Rock et al., supra.).

To examine if human TLRs might be involved in LPS-induced cellactivation, we first investigated the expression of TLRs in a variety ofimmune tissues. One of the TLRs, TLR2, was found to be expressed in alllymphoid tissues examined with the highest expression in peripheralblood leukocytes (FIG. 5 a). Expression of TLR2 is enriched inmonocytes/macrophages, the primary CD14-expressing and LPS-responsivecells. Interestingly, tLR2 is up-regulated upon stimulation of isolatedmonocytes/macrophages with LPS (FIG. 5 b), similar to what has beenreported for CD14 (Matsuura et al., Eur. J. Immunol. 22, 1663-1665[1992]; Croston et al., J. Biol. Chem. 270, 16514-16517 [1995]).

This result prompted us to determine, if TLR2 is involved inLPS-mediated cellular signaling. We engineered human embryonic kidney293 cells to express a version of TLR2 (gD-TLR2) containing anamino-terminal epitope-tag. A stable pool of clones as well as anindividual clone was isolated and shown to express a novel protein ofabout 105 kDa (FIG. 6 b), consistent with the predicted size of TLR2(˜89 kDa) and the presence of 4 potential sites for N-linkedglycosylation. We examined the response of 293 or 293-TLR2 cells and LBPby measuring the expression of a reported gene driven by the NF-κBresponsive enhancer of the E-selectin gene (Croston et al., supra).While neither LPS nor LBP treatment alone resulted in significant geneactivation, addition of both LPS and LBP resulted in substantialinduction of reporter gene activity in cells expressing TLR2, but not incontrol 293 cells (FIG. 6 a). Furthermore, using an electrophoreticmobility shift assay (EMSA), we found that LPS, in combination with LBP,induced NK-κB activity in TLR2 expressing cells (FIG. 6 c). The kineticsof LPS-induced NF-κB activity in 293-TLR2 cells resembled that ofmyeloid and nonmyeloid cells (Delude et al., J. Biol. Chem. 269,22253-22260 [1994]; Lee et al., Proc. Natl. Acad. Sci. USA 900,9930-9934 [1993]) in that nuclear localization of NF-κB is maximalwithin 30 minutes following exposure to LPS. Activation of NF-κB byLPS/LBP in 293-TLR2 cells does not require de novo protein synthesis,since pretreatment with cycloheximide (FIG. 6 c) or actinomycin D (notshown) does not inhibit NF-κB activation.

Both the membrane-bound form of CD14 (mCD14), which is present onmyeloid cells, and soluble CD14 (sCD14) which is present in plasma(Bazil et al., Eur. J. Immunol. 16, 1583-1589 [1986]), have been shownto enhance the responsiveness of cells to LPS. We observed that 293cells express little or no CD14 on their surface (data not shown).However, transient transfection of 293 cells which mCD14 increased thesensitivity and magnitude of TLR2-mediated LPS responsiveness (FIG. 6d).

The data presented above suggested that TLR2 might function as asignaling transducer for LPS. To examine the role of the intracellulardomain of TLR2 in mediating the LPS response, we determined if TLR2variants with C-terminal truncations of either 13 (TLR-M) or 141 aminoacids (TLR2-Δ2) could regulate the ELAM reporter in transientlytransfected 293 cells. We observed that both C-terminal truncationvariants were defective for activation of the reporter gene although wecould detect expression of these receptors at the cells surface by FACSanalysis (not shown) and by Western blot (FIGS. 7 c-7 d). The region ofthe intracellular domain deleted in TLR2-Δ1 bears striking similarity toa region of the IL-1R intracellular domain that is required froassociation with the IL-1R-associated kinase IRAK (Croston et al.,supra) (FIG. 7 b). We also demonstrated that the extracellular domain(ECD) of TLR2 is required for LPS-responsiveness in that a TLR2 variantin which the ECD of TLR2 was replaced with a portion of the ECD of CD4also failed to respond to LPS (FIGS. 7 a and 7 b).

LPS is a complex glycolipid consisting of the proximal hydrophobic lipidA moiety, the distal hydrophilic O-antigen polysaccharide region and thecore oligosaccharide that joins lipid A and O-antigen structures. Incontrast to the lipid A portion, there is a considerable diversity inthe O-antigen structures from different Gram-negative bacteria. Lipid Ais required for LPS responses, and treatments that remove the fatty acidside chains of lipid A inactivate LPS. We compared the potency of LPSprepared from various Gram-negative bacteria, as well as LPS which hadbeen “detoxified” by alkaline hydrolysis. We observed that LPS isolatedfrom Escherichia coli serotype LCD25 was nearly two orders of magnitudemore potent that the serologically distinct Escherichia coli 055:B5 LPSfor activating TLR2 (FIG. 8 a). LPS prepared from S. minnesota R595 LPSis also a potent inducer of TLR2 activity, while TLR2 failed to respondto “detoxified LPS”.

We examined if TLR2 binds LPS by determining if a soluble form of theTLR2 extracellular domain (TLR2-Fc) bound ³H-labeled LPS in an in vitroassay. We observed that ³H-LCD25 LPS bound the TLR2-Fc fusion protein,but did not bind either Fc alone, or fusion proteins containing the ECDof several other receptors (FIG. 8 b). This binding was specificallycompeted with cold LCD25 LPS but not with detoxified LPS. Preliminaryanalysis of the binding of LPS to TLR2-Fc suggests that the Kd isrelatively low (500-700 nM) and that the kinetics of binding are veryslow (data not shown). We speculate that other proteins, such as LBP,might act to enhance the binding of LPS to TLR2 in vivo, much like LBPacts to transfer LPS from its free, aggregated (micellar form) to CD14.This is consistent with our in vivo results showing that LBP is requiredto obtain a sensitive response of TLR2 to LPS (FIG. 6 a).

ILS treatment of macrophages leads to expression of a number ofinflammatory cytokines. Similarly expression of TLR2 in 293 cellsresulted in a >100 fold-induction of IL-8 mRNA in response to LPS/LBP,while detoxified LPS is inactive in this assay (FIG. 9).

These data suggest that TLR2 plays a sentinel role in the innate immuneresponse, the first line of defense against microbial pathogens. TLR2and CD14 are both expressed on myeloid cells, and their induction iscoordinately induced upon LPS treatment. Expression of TLR2 innon-myeloid cells confers LPS responsiveness to normally non-responsivecells by a mechanism that is dependent on LBP and is enhanced by theexpression of mCD14. LPS treatment of TLR2 expressing cells results inactivation of NF-κB and subsequent induction of genes that initiate theadaptive response such as IL-8 (FIG. 9). Our data suggest that TLR2participates in both sensing the presence of LPS and transmitting thisinformation across the plasma membrane because intact extracellular andintracellular domains are required for LPS responses. Moreover, a regionin the C-terminal tail of TLR2 that has homology to a portion of theIL-1R that is required for association with IRAK, is necessary for NF-κBactivation.

Drosophila Toll and the Toll related-receptor 18 Wheeler play andimportant role in the induction of antimicrobial peptides in response tobacteria and fungi, respectively. Medzhitov et al., supra. Genetic datahas implicated Spätzle as a ligand for Toll in dorsoventral patterningand has led to speculation that a homologue of Spätzle might beimportant for regulation of human TLRs in the immune response. Ourobservations that activation of TLR2 by LPS is not blocked bycycloheximide and that the extracellular domain of TLR2 is a lowaffinity receptor for LPS in vitro is consistent with a model in whichTLR2 participated in LPS recognition. Our data does not exclude thepossibility that other proteins (such as a Spätzle homologue) may modifythe response of TLR2 to LPS. We note that while extracellular domains ofTLR2 and Drosophila Toll both contain LRRs, they share less than 20%amino acid identity. Secondly, LRR proteins are Pattern RecognitionReceptors (PRRs) for a variety of types of molecules, such as proteins,peptides, and carbohydrates. Dangl et al., Cell 91, 17-24 (1997).Thirdly, the requirement for Spätzle in the Drosophila immune responseis less clear than that of Toll. Unlike defects in Toll, Spätzle mutantsinduce normal levels of the antimicrobial peptides Defensin and Attacinand are only partially defective in Cecropin A expression followingfungal challenge, and are not defective in activation of Dorsal inresponse to injury. Lemaitre et al., Cell 86, 973-983 (1996); Lemaitreet al., EMBO J. 14, 536-545 (1995).

As noted before, TLR2 is a member of a large family of humanToll-related receptors, including the three novel receptors (encoded byDNA40021, DNA42663, and DNA47361, respectively) specifically disclosedin the present application. The data presented in this example as wellas evidence for the involvement of TLR4 in activation of NF-κBresponsive genes, suggest that a primary function of this family ofreceptors is to act as pathogen pattern recognition receptors sensingthe presence of conserved molecular structures present on microbes,originally suggested by Janeway and colleagues (Medzhitov et al.,supra). The human TLR family may be targets for therapeutic strategiesfor the treatment of septic shock.

Example 12 In Situ Hybridization

In situ hybridization is a powerful and versatile technique for thedetection and localization of nucleic acid sequences within cell ortissue preparations. It may be useful, for example, to identify sites ofgene expression, analyze the tissue distribution of transcription,identify and localize viral infection, follow changes in specific mRNAsynthesis and aid in chromosome mapping.

In situ hybridization was performed following an optimized version ofthe protocol by Lu and Gillett, Cell Vision 1: 169-176 (1994), usingPCR-generated ³³P-labeled riboprobes. Briefly, formalin-fixed,paraffin-embedded human tissues were sectioned, deparaffinized,deproteinated in proteinase K (20 g/ml) for 15 minutes at 37° C., andfurther processed for in situ hybridization as described by Lu andGillett, supra. A [³³-P] UTP-labeled antisense riboprobe was generatedfrom a PCR product and hybridized at 55° C. overnight. The slides weredipped in Kodak NTB2 nuclear track emulsion and exposed for 4 weeks.

³³P-Riboprobe Synthesis

6.0 μl (125 mCi) of ³³P-UTP (Amersham BF 1002, SA<2000 Ci/mmol) werespeed vac dried. To each tube containing dried ³³P-UTP, the followingingredients were added:

2.0 μl 5× transcription buffer

1.0 μl DTT (100 mM)

2.0 μl NTP mix (2.5 mM: 10μ; each of 10 mM GTP, CTP & ATP+10 μl H₂O)

1.0 μl UTP (50 μM)

1.0 μl Rnasin

1.0 μl DNA template (1 μg)

1.0 μl H₂O

1.0 μl RNA polymerase (for PCR products T3=AS, T7=S, usually)

The tubes were incubated at 37° C. for one hour. 1.0 μl RQ1 DNase wereadded, followed by incubation at 37° C. for 15 minutes. 90 μl TE (10 mMTris pH 7.6/1 mM EDTA pH 8.0) were added, and the mixture was pipettedonto DE81 paper. The remaining solution was loaded in a Microcon-50ultrafiltration unit, and spun using program 10 (6 minutes). Thefiltration unit was inverted over a second tube and spun using program 2(3 minutes). After the final recovery spin, 100 μl TE were added. 1 μlof the final product was pipetted on DE81 paper and counted in 6 ml ofBiofluor II.

The probe was run on a TBE/urea gel. 1-3 μl of the probe or 5 μl of RNAMrk III were added to 3 μl of loading buffer. After heating on a 95° C.heat block for three minutes, the gel was immediately placed on ice. Thewells of gel were flushed, the sample loaded, and run at 180-250 voltsfor 45 minutes. The gel was wrapped in saran wrap and exposed to XAR,film with an intensifying screen in −70° C. freezer one hour toovernight.

³³P-Hybridization

Pretreatment of frozen sections The slides were removed from thefreezer, placed on aluminium trays and thawed at room temperature for 5minutes. The trays were placed in 55° C. incubator for five minutes toreduce condensation. The slides were fixed for 10 minutes in 4%paraformaldehyde on ice in the fume hood, and washed in 0.5×SSC for 5minutes, at room temperature (25 ml 20×SSC+975 ml SQ H₂O). Afterdeproteination in 0.5 μg/ml proteinase K for 10 minutes at 37° C. (12.5μl of 10 mg/ml stock in 250 ml prewarmed RNase-free RNAse buffer), thesections were washed in 0.5×SSC for 10 minutes at room temperature. Thesections were dehydrated in 70%, 95%, 100% ethanol, 2 minutes each.

Pretreatment of paraffin-embedded sections The slides weredeparaffinized, placed in SQ H₂O, and rinsed twice in 2×SSC at roomtemperature, for 5 minutes each time. The sections were deproteinated in20 μg/ml proteinase K (500 μl of 10 mg/ml in 250 ml RNase-free RNasebuffer; 37° C., 15 minutes)—human embryo, or 8× proteinase K (100 μl in250 ml Rnase buffer, 37° C., 30 minutes)—formalin tissues. Subsequentrinsing in 0.5×SSC and dehydration were performed as described above.

Prehybridization The slides were laid out in plastic box lined with Boxbuffer (4×SSC, 50% formamide)—saturated filter paper. The tissue wascovered with 50 μl of hybridization buffer (3.75 g Dextran Sulfate+6 mlSQ H₂O), vortexed and heated in the microwave for 2 minutes with the caploosened. After cooling on ice, 18.75 ml formamide, 3.75 ml 20×SSC and 9ml SQ H₂O were added, the tissue was vortexed well, and incubated at 42°C. for 1-4 hours.

Hybridization 1.0×10⁶ cpm probe and 1.0 μl tRNA (50 mg/ml stock) perslide were heated at 95° C. for 3 minutes. The slides were cooled onice, and 48 μl hybridization buffer were added per slide. Aftervortexing, 50 μl ³³P mix were added to 50 μl prehybridization on slide.The slides were incubated overnight at 55° C.

Washes Washing was done 2×10 minutes with 2×SSC, EDTA at roomtemperature (400 ml 20×SSC+16 ml 0.25M EDTA, V_(f)=4 L), followed byRNaseA treatment at 37° C. for 30 minutes (500 μl of 10 mg/ml in 250 mlRnase buffer=20 μg/ml), The slides were washed 2×10 minutes with 2×SSC,EDTA at room temperature. The stringency wash conditions were asfollows: 2 hours at 55° C., 0.1×SSC, EDTA (20 ml 20×SSC+16 ml EDTA,V_(f)=4 L).

Results

PRO285 (DNA40021)

The expression pattern of PRO285 (DNA40021) in human adult and fetaltissues was examined. The following probes were used, synthesized basedupon the full-length DNA40021 sequence:

Oligo 1: (SEQ ID NO: 27) GGA TTC TAA TAC GAC TCA CTA TAG GGC AAA CTC TGCCCT GTG ATG TCA Oligo 2: (SEQ ID NO: 28) CTA TGA AAT TAA CCC TCA CTA AAGGGA ACG AGG GCA ATT TCC ACT TAG

In this experiment, low levels of expression were observed in theplacenta and over hematopoietic cells in the mouse fetal liver. Noexpression was detected in either human fetal, adult or chimp lymph nodeand no expression was detected in human fetal or human adult spleen.These data are no fully consistent with Northern blot or PCR data,probably due to the lack of sensitivity in the in situ hybridizationassay. It is possible that further tissues would show some expressionunder more sensitive conditions.

PRO358 (DNA47361)

The expression pattern of PRO358 (DNA47361) in human adult and fetaltissues was examined. The following probes were used, synthesized basedupon the full-length DNA47361 sequence:

Oligo 1: (SEQ ID NO: 29) GGA TTC TAA TAC GAC TCA CTA TAG GGC TGG CAA TAAACT GGA GAC ACT Oligo 2: (SEQ ID NO: 30) CTA TGA AAT TAA CCC TCA CTA AAGGGA TTG AGT TGT TCT TGG GTT GTT

In this experiment, expression was found in gut-associated lymphoidtissue and developing splenic white pulp in the fetus. Low levelexpression was seen in the pALS region of normal adult spleen. Althoughall other tissues were negative, it is possible that low levels ofexpression could be observed in other tissue types under more sensitiveconditions.

Deposit of Material

The following materials have been deposited with the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md., USA (ATCC):

Material ATCC Dep. No. Deposit Date DNA40021-1154 209389 Oct. 17, 1997(encoding PRO285) DNA42663-1154 209386 Oct. 17, 1997 (encoding PR0286)DNA47361-1249 209431 Nov. 7, 1997

This deposit was made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposit will be made available byATCC under the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the culture of the depositto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trademarks to be entitledthereto according to 35 USC §122 and the Commissioner's rules pursuantthereto (including 37 CFR §1.14 with particular reference to 8860G 638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct deposited,since the deposited embodiment is intended as a single illustration ofcertain aspects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1. An isolated antibody which binds to a PRO286 polypeptide consistingessentially of amino acids 1 to 1041 encoded by SEQ ID NO:4.
 2. Theantibody of claim 1 wherein said antibody is a monoclonal antibody. 3.The antibody of claim 2 wherein said antibody is one of a chimeric,humanized, and human antibody.
 4. The antibody of claim 2 wherein saidantibody blocks binding of said polypeptide to one of a Gram-negativeand Gram-positive organism.
 5. An isolated antibody which specificallybinds to a PRO286 polypeptide consisting of amino acid residues 27 to825 of FIG. 3 (SEQ ID NO:3).
 6. The antibody of claim 5 wherein saidantibody is a monoclonal antibody.
 7. The antibody of claim 6 whereinsaid antibody is one of a chimeric, humanized, and human antibody.
 8. Anisolated antibody which binds to a PRO286 polypeptide consistingessentially of: (a) amino acids 1 to 1041 encoded by SEQ ID NO:4, or (b)amino acid residues 27 to 825 of SEQ ID NO:3; wherein said antibody isan agonist or an antagonist of NF-κB activation.
 9. The antibody ofclaim 8, wherein said antibody is an agonist of NF-κB activation. 10.The antibody of claim 8, wherein said antibody is an antagonist of NF-κBactivation.